bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4427: Role of Smurf2 in regulation of Smad anchor necessary for receptor activation (SARA) in TGFβ-receptor signaling
Authors: Lee Sanghyun , Di Guglielmo John M.
Published: July 2016

Non-small cell lung cancer (NSCLC) is a major form of lung cancer and is the leading cause of cancer mortality in the western world. Transforming growth factor β (TGFβ) deregulation leads to many human diseases: TGFβ is a tumor suppressor in normal lung epithelium; however, it switches roles and promotes lung cancer metastasis in cancer cells. TGFβ induces cell proliferation, migration and invasion in NSCLC cells.

The canonical pathway of TGFβ is initiated through binding of TGFβ ligand to transmembrane Ser/Thr kinase receptors, which propagate their signaling via receptor regulated R-Smads; proteins that function as intracellular effectors in the TGFβ pathway. Once activated, R-Smads form a complex with common (Co)-Smads that translocates into the nucleus to regulate transcriptional responses. Access of R-Smads to the activated receptor complex is regulated by an adaptor protein called Smad anchor for receptor activation (SARA). SARA facilitates the activation of Smads and allow efficient Smad signalling.

In addition to R-Smads and Co-Smads, inhibitory Smads (I-Smads) regulate TGFβ signalling. I-Smads block the signalling by competing against R-Smads for the association with TβR complex or by targeting receptors for ubiquitin-mediate degradation. I-Smads recruit the E3 ubiquitin ligases, Smad ubiquitination regulatory factors (Smurfs), to catalyze degradation of the receptor complex. The overall aim of the project is to characterize SARA in TGFβ receptor signalling and study the interaction of SARA with different proteins involved in the pathway, such as Smurf2 and Smad7.

We first examined how SARA, Smurf2 and Smad7 influence each other by performing a multi-combination transfection study. We also examined the interaction between SARA and Smurf2 through co-immunoprecipitation. We observed that the level of SARA decrease in the presence of Smurf2 and Smad7. In the presence of Smurf2 ligase inactive mutant and Smad7, the level of SARA is somewhat recovered. However, SARA does not directly interact with Smurf2. These data together suggest that SARA and Smurf2 influence each other in a very close manner. It also suggest that it could be a transient interaction. Characterizing SARA in TGFβ receptor signalling will provide us with possible drug targets for NSCLC.

Citation Format: Sanghyun Lee, John M. Di Guglielmo. Role of Smurf2 in regulation of Smad anchor necessary for receptor activation (SARA) in TGFβ-receptor signaling. [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 4427.
Abstract 4429: CCL20 regulation via TGFB /BMP signaling pathway in murine immortalized colonocytes and colonic epithelium
Authors: Brown Tasia D. , Means Anna , Freeman Tanner , Weaver Connie , Wilson Keith , Beauchamp. R. Daniel
Published: July 2016

TGFβ and BMP signaling through receptor-mediated activation of SMAD proteins is crucial for normal homeostasis in intestinal epithelium. Alterations in this pathway contribute to the development of colorectal cancers. To understand the role of these pathways in homeostasis of the colon we conditionally deleted loxP-flanked alleles of Smad4 in adult mouse colon. We used Lrig1CreERT allele to achieve greater than 80% SMAD4 gene deletion and a CK19CreERT allele for mosaic deletion in colonic epithelium. Our data indicates that SMAD4 activity regulates multiple interactions between intestinal epithelium cells and surrounding stroma and that deletion of SMAD4 in adult mouse colonic epithelial cells alters expression of cytokines, increases immune cell infiltration and predisposes mice to tumorigenesis. Increases in the chemokine CCL20 have been reported in CRC tumors beginning at the adenoma stage and it may regulate both Tregs and Th-17 cells in an active immune response. Luminex cytokine/chemokine array analysis revealed increased levels of CCL20 protein in colonic mucosa and RNAseq data revealed that when SMAD4 is knocked out in the epithelium there is an upregulation of CCL20 and CCR6 (receptor) mRNA in the epithelium and stroma, respectively. To further explore the regulation of CCL20, we used immortalized mouse colonocytes (IMC cells) to further determine how the TGFβ and BMP signaling pathways may be involved in CCL20 expression. We found that CCL20 mRNA expression was suppressed by TGFβ1 and BMP2 and increased by inhibitors of these pathways. The inhibition of CCL20 by the TGFβ and BMP2 pathways occurs even in the presence of a known strong inducer of CCL20, such as TNFα. We have further observed in mice induced to undergo deletion of SMAD4 in the intestinal epithelium that treatment with DSS results in 100% of mice developing mucinous adenocarcinomas in the distal colon. We conclude that ligand-dependent Smad4-mediated signaling provides important negative homeostatic regulation of CCL20 expression in colonic epithelium. Loss of Smad4 results in increased CCL20 expression and we are currently testing the hypothesis that CCL20 overexpression contributes to the inflammation induced carcinogenesis in this mouse model.

Citation Format: Tasia D. Brown, Anna Means, Tanner Freeman, Connie Weaver, Keith Wilson, R. Daniel Beauchamp. CCL20 regulation via TGFB /BMP signaling pathway in murine immortalized colonocytes and colonic epithelium. [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 4429.
Abstract 4431: Tandem profiling of 5-methyl and 5-hydroxymethylcytosine in glioblastoma identifies tumor-specific distribution of 5-hydroxymethylcytosine and associations with patient survival
Authors: Johnson Kevin C. , Houseman E. Andres , King Jessica E. , Fadul Camilo E. , Christensen Brock C.
Published: July 2016

Glioblastomas exhibit widespread molecular alterations including a highly distorted epigenome. Genome-wide assessment of epigenetic alterations to cytosines in glioblastoma has largely been limited to 5-methylcytosine (5mC). 5-hydroxymethylcytosine (5hmC) is an additional cytosine modification that is deregulated in cancer, though the extent and context of these alterations at single cytosine resolution remain unclear. Through parallel processing of DNA with bisulfite and oxidative bisulfite treatments on Illumina 450K arrays we resolved both 5mC and 5hmC in 30 glioblastomas. We developed and applied a novel technique for estimating 5mC, 5hmC, and unmethylated proportions from array data to glioblastoma tissues and compare with normal brain tissue. Genomic distribution of 5hmC was associated with features of transcription despite the glioblastoma genome being relatively depleted of 5hmC. When integrating 5mC and 5hmC data using a Gaussian finite mixture model approach, we observed significant associations between 5hmC levels and gene-sets involved in immune and RNA regulatory processes while high 5mC classes were associated with receptor-mediated processes and cancer gene-sets. Significant differences in patient survival were observed among classes of 5hmC obtained from a recursively partitioned mixture model. Gene expression across epigenetic enzymes including methyltransferases and TETs was also investigated for relation with patterns and levels of 5hmC and 5mC. Analyses of elevated 5hmC with alternative splicing events that may promote disease progression are underway. Together, our results provide a genome-wide map of 5hmC in glioblastoma, indicate that transcription patterns as well as disrupted processes are associated with 5hmC patterns, and highlight the potential clinical prognostic utility of 5hmC in cancer.

Citation Format: Kevin C. Johnson, E. Andres Houseman, Jessica E. King, Camilo E. Fadul, Brock C. Christensen. Tandem profiling of 5-methyl and 5-hydroxymethylcytosine in glioblastoma identifies tumor-specific distribution of 5-hydroxymethylcytosine and associations with patient survival. [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 4431.
Abstract 4443: Investigation of the promoter methylation status of the FHIT (fragile histidine triad) gene in larynx cancer
Authors: Demokan Semra , Akalin Kubra , Ozkoklesen Cansu , Ulusan Murat , Enver Necati , Dalay Nejat
Published: July 2016

Purpose: Development of carcinoma is associated with epigenetic modifications. Methylation of CpG islands in the promoter regions can participate in gene silencing and promote tumor aggressiveness. The Fragile histidine triad (FHIT) gene, localized on chromosome 3, is a tumor suppressor gene and the abnormal methylation of FHIT. The FHIT regulates the G2/M cell cycle check point and apoptotic pathwaysin various types of cancer. Inactivation of FHIT due to methylation has been shown in breast, cervical, gastric and colon carcinomas. In our study, we evaluated the FHIT gene promoter methylation in patients with larynx carcinoma (LC).

Experimental Design: Methylation of FHIT was investigated by bisulfite modification/methylation-specific polymerase chain reaction in tumors and matched normal tissue samples from 57 Turkish patients with LC.

Results: The promoter region of the FHIT was methylated in 96.49% and 29.82% of the primary tumors and the corresponding normal tissue samples, respectively [p = 0.0001, chi-square = 14.2105, OR(95%CI) = 64.7(14.4-296.07)]. A methylation rate (less than 10%) was observed in 3.51% (2/57) and 70.18% (40/57) of tumor and normal samples, respectively. Moderate methylation levels (10-25%) were found in 3.51% (2/57) of the tumors and 1.75% (1/57) of the adjacent normal tissues. High methylation levels (>25%) were observed in 92.98% (53/57) and 28.07% (16/57) in the tumor and normal tissues, respectively.

Conclusion: Methylation may play an important role in LC carcinogenesis. Although we found that there was significant association between the methylation levels of tumor and adjacent normal tissues, high methylation levels of the FHIT was observed in the tumors when compared to the levels in normal tissue. These results suggest that the methylation rates of FHIT gene may provide a suitable biomarker in LC carcinogenesis. Our study is still in progress to include a larger cohort of patients.

Citation Format: Semra Demokan, Kubra Akalin, Cansu Ozkoklesen, Murat Ulusan, Necati Enver, Nejat Dalay. Investigation of the promoter methylation status of the FHIT (fragile histidine triad) gene in larynx 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 4443.
Abstract 4444: DNA methylation age is accelerated in breast tissue of healthy women donors
Authors: Sehl Mary E. , Ganz Patricia A. , Horvath Steve
Published: July 2016

Background: Advanced age and cumulative estrogen exposure are well known risk factors for breast cancer. However, the mechanisms underlying the link between cellular senescence, chronic estrogen stimulation, increased cell cycling, and breast carcinogenesis are poorly understood. We examined epigenetic alterations that occur in the female breast with advancing age, and estrogen exposure.

Methods: We utilized specimens from 49 women healthy tissue donors from the Komen Tissue Bank who donated both breast tissue and peripheral blood at two time points spaced at least 2 years apart. Ages of the donors ranged from 18-65 years and they were grouped by menopausal status (27 pre- and and 22 post-menopausal) and parity (21 nulliparous). We assessed epigenome-wide DNA methylation levels (Illumina 450K platform) from DNA extracted from breast and blood tissue, and calculated tissue “DNA methylation age (DNAm age)” based on methylation levels at 353 CpGs that have been shown to strongly correlate with chronologic age across tissues. We examined the relationship between this outcome and age and variables related to cumulative estrogen exposure, including age at menarche, gravidity, parity, and menopausal status.

Results: DNAm age was highly correlated with chronological age in our sample (cor = 0.8, p<0.001), within both peripheral blood (cor = 0.94, p<0.001) and breast tissues (cor = 0.88, p<0.001). When we examine the residuals for each sample from our regression analysis of DNAm age versus chronological age, we find that age acceleration is significantly increased in breast relative to peripheral blood tissue (p<0.001). In a multivariate regression analysis, we found that earlier age at menarche (β = -0.62, p = 0.04) and higher gravidity (β = 0.95, p = 0.03) were associated with higher DNAm age.

Conclusions: We find that DNAm age is accelerated in breast tissue compared with peripheral blood cells in a population of healthy donors to the Komen Tissue Bank. We further note a significant association between DNAm age and early menarche, and rising number of pregnancies, suggesting that cumulative cell cycling with menstrual cycles and the tissue plasticity occurring with pregnancy may contribute to accelerated epigenetic age in the female breast.

Citation Format: Mary E. Sehl, Patricia A. Ganz, Steve Horvath. DNA methylation age is accelerated in breast tissue of healthy women donors. [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 4444.
Abstract 4456: Preliminary investigation of the promoter methylation status of BRCA1, BRCA2, TP53 genes of some breast cancer patients
Authors: Samuel Titilola A. , james babatunde ayo , HABEEB Muhammad , oshodi temitope , magbagbeola. olubunmi
Published: July 2016

Epigenetic alterations in BRCA1, BRCA2 and TP53, such as DNA methylation, may be an effective approach in diagnosis, prognosis and therapy in breast cancer. This study examines the DNA methylation signature of BRCA1, BRCA2 and TP53 in some breast cancer patients attending a Clinic in Lagos, Nigeria. In this study, 14 blood specimens with Invasive Ductal Carcinoma (IDC) were investigated. The samples were modified by Bisulfite modification, and then assessed by Methylation-Specific PCR (MSP). The methylation analysis indicates that 7(50%) of the 14 patients had a segment of the promoter region of BRCA1 gene methylated, 8 (57.14%) of BRCA2 methylated and 11(78.57%) of TP53 promoter region. This study provides preliminary evidence for the gene expression inactivation of BRCA 1, BRCA 2, and TP53 Genes in over 50% of the 14 patients with Stage 2 invasive ductal carcinoma who participated in this study.

Citation Format: Titilola A. Samuel, babatunde ayo james, Muhammad HABEEB, temitope oshodi, olubunmi magbagbeola. Preliminary investigation of the promoter methylation status of BRCA1, BRCA2, TP53 genes of some breast cancer patients. [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 4456.
Abstract 4460: Precision cancer risk diagnosis by accumulation of epigenetic alterations
Authors: Ushijima Toshikazu , Asada Kiyoshi , Maeda Masahiro , Nakajima Takeshi , Shimazu Taichi
Published: July 2016

Background and Aim: Aberrant DNA methylation is induced by chronic inflammation, and can be accumulated in normal-appearing tissues [reviewed in Ushijima and Hattori, Clin Cancer Res, 2:143, 2012]. Cross-sectional studies have shown that the degree of accumulation of aberrant DNA methylation was correlated with cancer risk, especially inflammation-associated cancers. In this study, we aimed to demonstrate that cancer risk can be predicted by an epigenetic cancer risk marker by a multicenter prospective cohort study.

Methods: We enrolled 826 patients with early gastric cancer, aged 40-80 years, who had undergone endoscopic resection. If a patient had active Helicobacter pylori (H. pylori) infection, the patient was enrolled after eradication of H. pylori. At the time of enrollment, one gastric biopsy was taken, and methylation levels of three preselected genes, miR-124a-3, EMX1 and NKX6-1, were measured by quantitative methylation-specific PCR. The patients were followed up annually by endoscopy to detect metachronous gastric cancer. To exclude cancers undetected at the time of enrollment, “authentic” metachronous gastric cancers were defined as those developed 1 year after enrollment.

Results: With a median follow-up of 2.97 years, 782 of 826 patients had at least one follow-up. Authentic metachronous gastric cancers developed in 66 patients: 29, 16 and 21 patients at 1-2, 2-3 and ≥3 years after the enrollment, respectively. The highest quartile of the miR-124a-3 methylation level had a significantly high HR in univariate analysis (95% CI) [2.17 (1.07 - 4.41); p = 0.032] and adjusted HR in multivariate analysis [2.30 (1.03 - 5.10); p = 0.042] [Asada et al, Gut, 64:388, 2015]. More importantly, with a median follow-up of 4.84 years, a multivariate analysis yielded a larger HR [2.79 (1.34 - 5.81)] with a smaller p value (0.0061).

Conclusions: This study, for the first time, demonstrated the usefulness of an epigenetic cancer risk marker by a multicenter prospective cohort study. DNA methylation accumulated in normal-appearing tissues was considered to be highly useful for precision cancer risk diagnosis.

Citation Format: Toshikazu Ushijima, Kiyoshi Asada, Masahiro Maeda, Takeshi Nakajima, Taichi Shimazu. Precision cancer risk diagnosis by accumulation of epigenetic alterations. [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 4460.
Abstract 4462: Cytotoxic effects of a novel BRD4 inhibitor in uveal melanoma cells with Gnaq/11 mutations
Authors: Ambrosini Grazia , Schwartz Gary K.
Published: July 2016

Uveal melanoma (UM) is an aggressive intraocular malignancy with high tendency to metastasize to the liver. The majority of UM harbor activating mutations in the G proteins Gnaq/11, and amplification of the oncogene c-Myc. Currently available drugs have shown limited response in patients with UM and there is an urgent need for new effective therapies. Recent findings from our laboratory demonstrated that UM cells with Gnaq/11 mutations are dependent on BRD4 activity. BET proteins are epigenetic readers involved in chromatin remodeling and transcriptional regulation. BRD4 inhibitors consist of small molecules that competitively displace BRD4 from acetylated histones, resulting in the suppression of the MYC transcriptome and other BRD4-dependent genes. Here, we report that a novel clinical BRD4 inhibitor from Plexxikon (Berkeley, CA), PLX51107, induces high cytotoxic activity in UM cell lines at nanomolar concentrations. In cells with Gnaq/11 mutations, PLX51107 showed an IC50 of 250nM, similar to the pre-clinical compound JQ1. In contrast, PLX51107 did not have a significant effect (IC50>3000nM) in cells without the mutations. PLX51107 induced marked apoptosis in Gnaq/11 mutant cells, as detected by Annexin V staining and induction of cleaved PARP. It also suppressed the expression of BRD4 targets, i.e c-Myc, Rad51, and induced the pro-apoptotic protein Bim. In mouse xenograft models of UM PLX51107 significantly inhibited tumor growth. Since the MAPK pathway plays a major role in UM, we explored the combination of PLX51107 with a MEK inhibitor, and found increased activity in vitro and in vivo. All together, these observations support the idea that this clinical BRD4 inhibitor represents a novel therapeutic intervention against UM with Gnaq/11 mutations, and clinical development is planned.

Citation Format: Grazia Ambrosini, Gary K. Schwartz. Cytotoxic effects of a novel BRD4 inhibitor in uveal melanoma cells with Gnaq/11 mutations. [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 4462.
Abstract 4471: Overcoming the resistance to the anti CD20 treatments in chronic lymphocytic leukaemia
Authors: Scialdone Annarita , Kofoed Damm Jesper , Gullberg Urban , Drott Kristina
Published: July 2016

Chronic lymphocytic leukaemia is the most common leukaemia in the Western countries. Treatment of CLL and other lymphomas is based on cytostatic drugs in combination with rituximab or other monoclonal antibodies targeting the B-cell surface marker CD20. Although the CD20-antibodies have revolutionized the therapeutic perspectives of indolent B-cell lymphomas, the treatment of CLL with CD20-antibodies is often inefficient, a feature which has been explained by the inherently low CD20 expression in CLL. It has been shown previously that CD20 is epigenetically regulated in some lymphoma cell lines and that some histone deacetylase inhibitors (HDACi) can increase CD20 expression in B-cell lymphomas both in vitro and in vivo. Therefore, we utilized the HDACi valproate to upregulate CD20 both in CLL cell lines and in patients.Two CLL patients with del13q were treated with valproate at serum levels around 800 μM for three days in three 21-day cycles. CD20 expression was analyzed by Real time-PCR and flowcytometry on the B-CLL cells isolated from peripheral blood.In addition, the valproate-induced histone modifications and changes in repressor/activator complexes of the CD20 promoter were investigated in the CLL cell lines and in patients.In the patient 1, CD20 expression was increased 2 times after 2 weeks of treatment, suggesting long-term effects by valproate. Neither the mRNA stability nor the protein stability seems to be affected by Valproate, suggesting transcriptional effects. However, no effects were observed in the patient 2. In contrast to patient results, Valproate increases CD20 transcript and protein levels by 2,5 times in the CLL-cell line I83-E95 after 48 hours. Here, the activating histone mark, acetylated H3K9, is enriched on the CD20 promoter suggesting valproate-related effects on the epigenome. Our study will continue the characterization of valproate-related effects on the epigenetic signatures of the CD20 promoter in CLL cells in vitro and in vivo, dissecting the mechanisms underling low CD20 expression in CLL.

Citation Format: Annarita Scialdone, Jesper Kofoed Damm, Urban Gullberg, Kristina Drott. Overcoming the resistance to the anti CD20 treatments in chronic lymphocytic leukaemia. [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 4471.
Abstract 4477: Loss of EZH2 accelerates STAT5 loss mediated fatty liver and cancer development through non-methyltransferase function
Authors: Bae Woo Kyun , Shim Hyun Jeong , Cho Sang Hee , Chung Ik-Joo , Kim Kyung Keun
Published: July 2016

Background: The molecular mechanisms underlying the development of fatty liver and hepatocellular carcinoma are not fully understood. Loss of STAT5 from liver tissue results in hepatosteatosis and hepatocellular carcinoma (HCC). Enhancer of zeste homolog 2 (EZH2) mediates epigenetic silencing of gene expression and is frequently up-regulated in human HCC. STAT5 recruits EZH2, which represses a substantial subset of genes regulated by STAT5 during B lymphopoiesis. However, it is not clear if EZH2 can affect STAT5-target genes in liver.

Methods: To investigate the role of STAT5 and EZH2 in liver, mice were generated that carried EZH2fl/fl alleles, STAT5fl/fl alleles, EZH2fl/flSTAT5fl/fl alleles and an Alb-Cre transgene. Lipid analysis in serum and liver, and RNA-seq were examined at 3 & 8 months of age. Mice were also examined by chronic 3 months injection by carbon tetrachloride (CCl4).

Results: The deletion of STAT5 in hepatocyte caused fatty liver and increased cholesterol in serum and liver. The deletion of EZH2 did not make a difference in H3K27me3 expression, and lipid in serum and liver. However, combined loss of EZH2 and STAT5 caused severe fatty liver, increase triglyceride and the ratio liver/body weight compare to STAT5 KO mice (the ratio; 0.097 vs. 0.058, p < 0.05). In response to 3 months treatment with CCl4, combined loss of EZH2 and STAT5 showed accelerated development of hepatocellular carcinoma at 12 months of age. Transcriptome analysis identified the expression of several genes was significantly increased (cd36, pparγ, cyp4a14, lipoprotein lipase, fasn) and decreased (nox4, bbc3, bcl2l1, cdkn2b) which were well known as STAT5 target genes in combined loss of EZH2 and STAT5.

Conclusions: This work demonstrates that loss of EZH2 in liver affects the expression of STAT5 target genes and accelerates STAT5 loss mediated fatty liver and hepatocellular carcinoma development through non-methyltransferase function.

Citation Format: Woo Kyun Bae, Hyun Jeong Shim, Sang Hee Cho, Ik-Joo Chung, Kyung Keun Kim. Loss of EZH2 accelerates STAT5 loss mediated fatty liver and cancer development through non-methyltransferase function. [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 4477.
Abstract 4478: EGFR tyrosine kinase inhibitors overcome resistance to chemotherapy in malignant pleural mesothelioma
Authors: Staumont Bernard , Costa Chrisostome , Vandermeers Fabian , Neelature Sriramareddy Sathya , Mascaux Céline , Scherpereel Arnaud , Duysinx Bernard , Louis Renaud , Delvenne Philippe , Hubert Pascale , Willems Luc
Published: July 2016

Background: Malignant pleural mesothelioma (MPM) is a cancer of the pleura mainly caused by exposure to asbestos fibers. Current treatments are unsatisfactory due to intrinsic chemoresistance of the tumor. We hypothesized that chemoresistance was due to epigenetic errors and evaluated the ability of HDAC inhibitors to improve treatment efficacy. We previously showed that valproic acid (VPA) improves the first line regimen of MPM both in vitro and in vivo (Vandermeers et al, 2009, Clinical Cancer Research 15: 2818). A clinical trial also demonstrated that VPA in combination with doxorubicin increases the response rate of second line patients (Scherpereel et al, 2011, European Respiratory Journal 37:129).

Methods: Transcriptomic profiling was performed by microarray analyses (Agilent). Gene expression was validated by quantitative RT-qPCR. Modulation of TGFα expression was performed by shRNA interference and transfection of a cDNA vector. Onset of apoptosis was assessed with the Annexin V assay.

Results: To evaluate the mechanisms associated with the response to chemotherapy, we compared two types of MPM cell lines (M14K and H28) characterized by a difference in sensitivity to doxorubicin+VPA. Microarray analyses and bioinformatic modeling of gene expression profiles revealed the most relevant candidate genes associated with sensitivity or resistance to this regimen. Among these, TGFα expression was associated with resistance to doxorubicin + VPA in a series of MPM cell lines. Silencing of TGFα by RNA interference in H28 cells correlated with a significant increase in apoptosis. On the other hand, overexpression of TGFα desensitized M14K cells to doxorubicin+VPA -induced apoptosis. Since TGFα interacts with the EGF receptor, we evaluated pharmacological inhibition using EGFR tyrosine kinase inhibitors (erlotinib and gefitinib) and the dual HDAC/EGFR inhibitor CUDC-101. As predicted, these TKI inhibitors improved efficacy of doxorubicin+VPA.

Conclusions: Our data demonstrate that TGFα is involved in resistance of MPM to chemotherapy and that TKI inhibitors overcome resistance to second line regimen.

Citation Format: Bernard Staumont, Chrisostome Costa, Fabian Vandermeers, Sathya Neelature Sriramareddy, Céline Mascaux, Arnaud Scherpereel, Bernard Duysinx, Renaud Louis, Philippe Delvenne, Pascale Hubert, Luc Willems. EGFR tyrosine kinase inhibitors overcome resistance to chemotherapy in malignant pleural mesothelioma. [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 4478.
Abstract 4480: Blood epigenetic age may predict cancer incidence and mortality
Authors: Zheng Yinan , Joyce Brian T. , Colicino Elena , Liu Lei , Zhang Wei , Dai Qi , Shrubsole Martha J. , Kibbe Warren A. , Gao Tao , Zhang Zhou , Jafari Nadereh , Schwartz Joel , Baccarelli Andrea A. , Hou Lifang
Published: July 2016

Biological measures of aging are important for understanding age-related cancers as the population ages. Since the epigenome is closely related to aging, epigenetics may help predict these and other age-related diseases. We aimed to prospectively examine whether blood Δage (the discrepancy between epigenetic and chronological age) can predict cancer incidence/mortality. In a prospective cohort (the Normative Aging Study), Δage and its rate of change over time were calculated using Illumina Human Methylation 450K array data in 834 blood leukocyte samples longitudinally collected from 442 participants free of cancer at the blood draw. About 3-5 years before cancer onset or death, Δage was associated with cancer risks in a dose-responsive manner (test for trend P = 0.02) and time-dependent Cox models showed a one-year increase in Δage was associated with all-cancer incidence (HR (hazard ratio): 1.06, 95% CI: 1.02-1.10) and mortality (HR: 1.17, 95% CI: 1.07-1.28). Participants with smaller Δage and decelerated epigenetic aging over time had the lowest risks of cancer incidence (log-rank P = 0.003) and mortality (log-rank P = 0.02). Spline analysis suggested Δage was associated with cancer incidence in a ‘J-shaped’ manner, and with cancer mortality in a time-varying manner. We conclude that blood epigenetic age may mirror epigenetic abnormalities related to cancer early development or the immune response to it. Those with older epigenetic age relative to their chronological age have an elevated risk of cancer events within 3-5 years. Thus, epigenetic age could potentially serve as a novel, minimally invasive biomarker for cancer prediction.

Citation Format: Yinan Zheng, Brian T. Joyce, Elena Colicino, Lei Liu, Wei Zhang, Qi Dai, Martha J. Shrubsole, Warren A. Kibbe, Tao Gao, Zhou Zhang, Nadereh Jafari, Joel Schwartz, Andrea A. Baccarelli, Lifang Hou. Blood epigenetic age may predict cancer incidence and mortality. [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 4480.
Abstract 4482: Expression profiling of SETD family genes in breast cancer patients
Authors: Matos Neto Joao N. , do Amaral Rabello Doralina , Pittella-Silva Fabio
Published: July 2016

Epigenetic alterations have recently been recognized to play an important role in the progression of many tumor types. Protein methyltrasferases are among the most relevant epigenetic effectors. Dysregulation in the expression or activity of these enzymes is among the major epigenetic alterations involved in carcinogenesis. Methyltransferases of the SETD family share a SET domain which is involved in the methylation of lysine residues on histones and non-histone proteins. So far, little is known about the relation of SETD family genes with mammary carcinogenesis. In the present study we evaluated the relative expression of 10 SETD family genes in 18 breast tumor samples and non-tumor breast tissues from patients with breast cancer by real time qPCR. The comparative expression profile of each gene was correlated with the most relevant clinicopathologic prognostic information available from patients. We found a significant increase in the expression of SETMAR in tumor samples compared with the normal breast tissues. In addition, triple negative tumor subtype (n = 7), when compared with a pool of normal tissue samples (n = 13) presented an increased relative expression of SETMAR. Interestingly, SETD8 was 10 times overexpressed when the angiolymphatic invasion (n = 8) was present compared to the absence of this feature (n = 10). Patients with strong axillary tumor burden defined as the presence of at least 10 axillary lymph nodes with cancer (n = 6), showed an increased relative expression of SETD5 and SETD8 genes when compared to the absence of this feature (n = 12). Evaluating the expression on paired samples (normal tissue and tumor tissue from the same patient (n = 7), we observed an increased relative expression of SETD1B. Also in the paired samples, we found a strong correlation among the expression of the genes SETD1B, SETD5, SETD8 and SETMAR in tumor samples with no correlation in normal tissues. Furthermore, immunohistochemical analysis revealed a stronger expression of SETD5 and SETMAR in carcinomas in situ when compared to normal tissues and invasive tumors. Taken together, these data points to a likely role of SETMAR and SETD1B genes in mammary carcinogenesis, with relevant clinical implications and with the possible involvement of SETD5 and SETD8 genes in more advanced stages of the disease. The correlation on the expression of these genes with relevant clinical prognostic factors in breast cancer opens the way to exploit them not only as therapeutic targets, but also as new prognostic markers.

Keywords: SETD gene family, expression profile, breast cancer

Citation Format: Joao N. Matos Neto, Doralina do Amaral Rabello, Fabio Pittella-Silva. Expression profiling of SETD family genes in breast cancer patients. [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 4482.
Abstract 4501: Immunophenotypic identities of clinical samples have the potential to correlate with overall survival in cytogenetically normal AML patients
Authors: Gaj Pawel , Nowis Dominika , Volinia Stefano
Published: July 2016

Tumor infiltrating lymphocytes (TILs) have been described to have great potential to modulate course of the disease. Capacity to eliminate cancer cells primarily depends on the interplay between tumor and its microenvironment. Despite the great interest which this topic gathered over last decades the role of immune cells interaction with cancer has hardly been studied in acute myeloid leukemia patients.

Multitude of patient-specific factors, as well as a great repertoire of different kinds of tumor-infiltrating immune cells, potentially affecting disease progression should be the motivation to investigate implication of tumor immune cells interplay in possibly maximized cohorts of patients. Due to technical reasons the traditional microscopy-based techniques do not allow for the desired high-throughput approach.

In the present study we demonstrate an inter-platform-compatible method which allowed us to predict the gene expression component involvement of 37 cell populations representing different stages of hematopoietic linage differentiation. The predictions were done for an extensive set of peripheral blood mononuclear cells (PBMC) and bone marrow aspirate material sampled from cytogenetically normal (CN) AML cases.

Our results nicely confirmed poor survival prognosis observed for the patients demonstrating high contribution of the CD34+, CD38- hematopoietic stem cells in their bone marrow samples HR = 2.05, 95%CI = 1.22-3.43, p-value = 6.73E-03. We also show that a specific Mature NK cell_CD56+ CD16+ CD3- component found in gene expression signatures of bone marrow samples correlated independently with significantly worse survival in CN AML patients HR = 2.56, 95%CI = 1.49-4.42, p-value = 7.03E-04.

Same analysis carried for the CN AML PBMC samples indicated independent positive effects for the predicted involvement of the Erythroid_CD34+ CD71+ GlyA- and Hematopoietic stem cell_CD133+ CD34dim cell populations in The Cancer Genome Atlas (TCGA) CN AML cohort.

Acknowledgements

The research was supported by a grant from European Commission 7th Framework Programme: FP7-REGPOT-2012-CT2012-316254-BASTION, a grant from the Ministry of Science and Higher Education of Poland [IP2012 048172 (DN)], and a grant from the National Science Centre Poland [2013/10/E/NZ5/00778 (DN)].

Citation Format: Pawel Gaj, Dominika Nowis, Stefano Volinia. Immunophenotypic identities of clinical samples have the potential to correlate with overall survival in cytogenetically normal AML patients. [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 4501.
Abstract 4502: Molecular impact of in vitro exposure to electronic cigarette vapor in human bronchial epithelium
Authors: Moses Elizabeth , Wang Teresa , Jackson George R. , Corbett Sean , Drizik Eduard , Brooks Daniel , O’Connor George , Perdomo Catalina , Dubinett Steven , Hayden Patrick , Lenburg Marc E. , Spira Avrum
Published: July 2016

Electronic cigarettes (ECIGs) are an emerging alternative tobacco product thought by some to potentially be safer than traditional tobacco cigarettes (TCIGs). Despite the increasing prevalence of ECIG use, few studies have evaluated the potential physiological effects of ECIG exposure. In this study we aimed to determine the global gene expression effects of ECIG exposure on bronchial epithelium in vitro. Human bronchial epithelial cells (HBECs) grown at Air Liquid Interface (ALI) were exposed to TCIG smoke and ECIG vapor derived from tobacco or menthol flavored products with and without nicotine. We identified a number of gene expression alterations that were induced by both ECIG and TCIG exposure as well as a novel set of changes uniquely induced by ECIG exposure. ECIG exposure induced the expression of genes involved in oxidative and xenobiotic stress pathways and increased the production of reactive oxygen species, similar to, but generally lower in magnitude than, the effects of TCIGs. Furthermore, TCIG and ECIG exposure both decreased the expression of genes involved in cilia assembly and movement, suggesting that the integrity of the bronchial epithelium is concordantly impaired by both exposures. We additionally identified a number of ECIG-specific cell cycle and cell division pathway changes. Finally, we observed that ECIG-induced changes were dependent on both flavor and nicotine content. Together, these results indicate that ECIG vapor can induce cellular stress and molecular alterations within airway epithelium that share similarities with the effects of TCIG smoke. Based on these findings, further studies are warranted to determine whether ECIG use will lead to similar deleterious health outcomes as those caused by TCIGs.

Citation Format: Elizabeth Moses, Teresa Wang, George R. Jackson, Sean Corbett, Eduard Drizik, Daniel Brooks, George O’Connor, Catalina Perdomo, Steven Dubinett, Patrick Hayden, Marc E. Lenburg, Avrum Spira. Molecular impact of in vitro exposure to electronic cigarette vapor in human bronchial epithelium. [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 4502.
Abstract 4505: Pan-cancer analysis of fusion gene druggability and neoantigen potential
Authors: Edgren Henrik , Ruusulehto Anja , Ojala Kalle , Laaksonen Maria
Published: July 2016

Fusion genes are a well known type of oncogenic mutation, in which a genomic rearrangement, such as e.g. a translocation, inversion or deletion, joins together parts of two genes, which normally are not located adjacent to each other. In the context of cancer driver mutations, the result of this rearrangement is a novel chimeric gene with oncogenic potential. While fusion genes have been known longest in hematological malignancies, over the last years they have been shown to occur in nearly all types of cancers studied. Notable examples include ETS-family fusions, such as TMPRSS2-ERG, in prostate cancer.

In previous work, we have used our FusionSCOUT fusion gene detection pipeline to analyze over 8000 cancer samples from 28 different TCGA cancer types. In this study, we extend this target discovery to further characterize the genomic alteration contexts in which gene fusions occur, both by examining the copy number states at inferred rearrangement breakpoints, as well as by correlating the numbers of fusion genes identified in tumors with the level of genomic instability in the tumors. We also analyze the frequency at which different druggable protein domains occur in fusion genes, comparing these to the expected random distribution. Finally, we predict, for each tumor, the neoantigen potential of the identified fusion proteins and compare this to the same predictions for simple somatic mutations, in order to characterize the contribution of fusion proteins to neoantigen load.

In summary, we extend our pan-cancer fusion gene identification analysis with a detailed characterization of the genomic alteration contexts at which fusion genes occur. We also report on the drug treatment potential of fusion genes, both in terms of small molecule drugs, as well as highlight the degree to which fusion gene derived chimeric proteins should be taken into account when estimating the neoantigen load of tumors.

Citation Format: Henrik Edgren, Anja Ruusulehto, Kalle Ojala, Maria Laaksonen. Pan-cancer analysis of fusion gene druggability and neoantigen potential. [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 4505.
Abstract 4513: A prognostic model for pulmonary carcinoid tumors based on large chromosomal alterations
Authors: Leventakos Konstantinos , Mansfield Aaron S. , Murphy Stephen J. , Johnson Sarah H. , Kerr Sarah E. , Aubry Marie Christine , Vasmatzis George , Molina Julian R.
Published: July 2016

PURPOSE: Previous mutational analyses have failed to identify a predictive or prognostic marker for pulmonary carcinoid tumors (PCT). The heterogeneity of PCTs coupled with a lack of detailed information about the tumor biology, impedes patient stratification into groups based on tumor phenotypes or treatment response. We sought to use high-throughput next-generation sequencing to improve prognostication.

METHODS: We stratified 37 patients with resected PCTs as low risk (n = 17) and high risk (n = 20) based on a 5 year long follow up for recurrence. Patients with no recurrence within 5 years from initial treatment were deemed low risk. Macrodissection was followed by genomic DNA isolation and next-generation sequencing was performed using an Illumina Mate Pair library protocol. Sequence reads were mapped to the human genome, and primers spanning the fusion junctions were used for validation polymerase chain reaction.

RESULTS:

Carcinoids commonly harbor rearrangements (median 3.5, range 1-167). Large chromosomal gains or losses were found in 24 of the cases (64.8%). We developed a prognostic score that included the total amount of genetic alterations (deletions and translocations). ROC analysis revealed an AUC of 0.76. High risk patients had a mean score of 20 and low risk patients had a mean score of 5.45 (p value = 0.03, Welch Two Sample t-test). Further analysis of the specific genetic alterations harbored by high and low risk PCTs and correlation with the pathologic and immunohistochemical information is currently underway.

CONCLUSION:

Large chromosomal rearrangements and aneuploidy portend a poor prognosis for patients with PCTs. Mate Pair sequencing of PCTs provides valuable prognostic information for this highly heterogeneous group of cancers.

Citation Format: Konstantinos Leventakos, Aaron S. Mansfield, Stephen J. Murphy, Sarah H. Johnson, Sarah E. Kerr, Marie Christine Aubry, George Vasmatzis, Julian R. Molina. A prognostic model for pulmonary carcinoid tumors based on large chromosomal alterations. [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 4513.
Abstract 4524: A rational approach for the discovery of inhibitors of NSD2 for the treatment of multiple myeloma
Authors: Fromond Claudia , Espanel Xavier , Estevez Severine , Adarbes Vanessa , Bocart Stephanie , Loillier Bruno , Soude Anne , Urban Nathalie , Bell Frederic , Masson Philippe , Boubia Benaïssa , Broqua Pierre , Montalbetti Christian
Published: July 2016

Multiple myeloma (MM) is a plasma cell malignancy which accounts for approximately 10% of hematologic malignancies. Despite the introduction of new therapeutic agents, MM remains incurable and nearly all patients ultimately relapse. About 20% of MM are due to a chromosomal translocation t(4;14) leading to overexpression of the NSD2 histone methyltransferase. NSD2 catalyzes dimethylation of lysine 36 on histone H3 (H3K36me2) and is associated with transcriptionally active regions. Several studies have shown that in MM harboring the translocation t(4;14), oncogenic programming is dependent on the methyltransferase activity of NSD2. Thus, NSD2 inhibitors are potential therapeutics for this devastating cancer

Using the AlphaLisa™ technology, we screened 240,000 compounds coming from our proprietary library. The assay was based on the detection of H3K36me2 on nucleosome by a specific antibody. False positives hits were removed by a technological counterscreen, and compounds with redox or metal contamination activity were excluded. For confirmation purposes, an orthogonal counter-screen based on 3H SAM incorporation was performed. The IC50 obtained for one of the best compounds across the AlphaLisa and 3H SAM incorporation are very similar, showing values of 0.15 μM and 0.29 μM respectively for compound 1.

Binding affinities measured by MST and SPR for compound 1 were consistent with the IC50 observed in the biochemical assays KD = 0.19 μM for MST and 1.7 μM by SPR. This compound was also shown to be a good binder by NMR (STD), and further experiments are planned to elucidate the selectivity and MOA of this series.

To our knowledge, no NSD2 inhibitor have been identified to date despite several screening efforts performed by other groups. We believe that our hits are promising starting points to generate potent and selective NSD2 inhibitors.

Citation Format: Claudia Fromond, Xavier Espanel, Severine Estevez, Vanessa Adarbes, Stephanie Bocart, Bruno Loillier, Anne Soude, Nathalie Urban, Frederic Bell, Philippe Masson, Benaïssa Boubia, Pierre Broqua, Christian Montalbetti. A rational approach for the discovery of inhibitors of NSD2 for the treatment of multiple myeloma. [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 4524.