bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2391: Integrated analysis of methylation and expression profile of telomere-related genes in breast cancer patients
Authors: Heng Jianfu , Guo Xinwu , Tang Lili , Zhang Fan , Peng Limin , Chen Ming , Luo Xipeng , Xu Xunxun , Wang Shouman , Wang Jun
Published: July 2017

Telomeres at the ends of chromosomes are critical in maintaining the integrity and stability of the genome. Aberrant telomere or telomerase dysfunction participates in tumorgenesis. A majority of human cancers exhibit critically aberrant telomere length, suggesting that tumors can arise from genetically instable cells with dysfunctional telomeres. Many genes are involved in the complex regulatory mechanisms of telomere length and telomerase activity. In addition, the methylation and expression pattern for most of telomere related genes in breast cancer are still unknown. Using microfluidic-PCR based target enrichment and next-generation bisulfite sequencing technology, we explored the promoter methylation profile of 29 telomere-related genes in 184 breast cancer patients with paired tumor and matched normal tissues. The average methylation level was significantly higher in tumor (8.13%) than that in matched normal tissues (7.08%) (P = 4.30E-21). Four genes showed significant hyper-methylation in the breast tumor tissues. All of these 4 genes are annotated with potential TFBSs in the promoter regions. In subtype analysis, RAD51D showed significant methylation difference among four subtypes. In analysis of the association between methylation and clinicopathologic characteristics, TERC showed significant difference between ER+/ER- tumors. The expression profile of the 4 significant hyper-methylated genes was explored in the same cohort using qPCR method. All of them showed significantly lower expression in breast tumor tissues compared with the matched normal tissues. Two genes showed significant and negative cis correlation between methylation and gene expression. These results were also validated in the TCGA database. The 4 genes panel showed a good performance in predicting breast cancer with ROC analysis. In summary, our results revealed the methylation pattern of telomere related genes in breast cancer and illustrated the epigenetic regulatory mechanism on expression of aberrant methylated genes. Our study provides a novel panel of telomere related genes which may be a valuable diagnostic biomarker for breast cancer prediction.

Note: This abstract was not presented at the meeting.

Citation Format: Jianfu Heng, Xinwu Guo, Lili Tang, Fan Zhang, Limin Peng, Ming Chen, Xipeng Luo, Xunxun Xu, Shouman Wang, Jun Wang. Integrated analysis of methylation and expression profile of telomere-related genes in breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2391. doi:10.1158/1538-7445.AM2017-2391
Abstract 2394: Subpopulation of infiltrating lymphocyte and MGMT methylation in brain tumor prognosis
Authors: Castaneda Carlos , Castillo Miluska , Belmar-Lopez Carolina , Casavilca Sandro , Bernabe Luis Angel , Vidaurre Tatiana
Published: July 2017

Background: Clinicopathological features including MGMT methylation have been correlated to prognosis in Glioblastoma (GBM). Tumor infiltrating lymphocytes (TILs) are predictive of clinical outcome in different malignancies. We evaluated association between clinicopathological features including MGMT methylation and IHC infiltrating immune cell subpopulations in 43 resected GBM cases who came from 2008 to 2013.

Methods: A prospective evaluation of TIL (CD3, CD4, CD8 and CD20) and macrophages (CD68 and CD163) through H&E was performed by a pathologist and evaluated Immune cell subpopulations was evaluated through scanning, and software analysis (BX63 Olympus and TissueMorph-Visionpharm) with pathologist support. MGMT evaluation was prospectively performed by bisulphyte PCR test in paraffin samples.

Results: Median age was 47.9 y, men (51.2%), Karnofsky scale 90% (62.8%), median tumor size of 5.1 cm, and partial/ complete tumor resection (22/21). Methylation MGMT test was performed in 31 cases and 17 showed un- methylated, 4 partial methylated and 10 methylated. TILs evaluation by HE showed low- intensity (29 cases), middle- intensity (1) and high- intensity level (9). Pattern of distribution was focal (9 cases), multi- focal (13) and diffuse (17). Median CD3, CD4, CD8, CD20, CD68 and CD163 were 1.6%, 0.032%, 1.6%, 0.03%, 9.1% and 2.2% (regarding total cells), respectively. Higher CD163 level was associated to male (p=0.03) and larger size (p=0.05). Higher levels of CD8 was associated to larger size (p=0.05). Higher CD8 and CD20 was associated to unmethylated MGMT (p=0.041 and 0.013). Median follow- up was 14 months. Only total resection (p<0.05) and MGMT methylation (p<0.05) was associated to outcome. Lymphocyte infiltration (H&E) was not associated to outcome: Lymphocyte intensity (p=0.703), distribution (p=0.61). Longer OS was associated to lower CD4 levels (p=0.01).

Conclusion: Tumor Infiltrating immune cell are not common in GBM, CD8 and CD20 was associated to unmethylation, and CD4 and MGMT methylation were associated to prognosis.

Citation Format: Carlos Castaneda, Miluska Castillo, Carolina Belmar-Lopez, Sandro Casavilca, Luis Angel Bernabe, Tatiana Vidaurre. Subpopulation of infiltrating lymphocyte and MGMT methylation in brain tumor prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2394. doi:10.1158/1538-7445.AM2017-2394
Abstract 2397: Epigenetic silencing in clear renal cell carcinoma: KEAP1 promoter hypermethylation
Authors: Fabrizio Federico Pio , Costantini Manuela , Copetti Massimiliano , Torre Anna Maria la , Sparaneo Angelo , Fontana Andrea , Poeta Maria Luana , Gallucci Michele , Sentinelli Steno , Graziano Paolo , Parente Paola , Pompeo Vincenzo , Salvo Laura De , Simone Giuseppe , Papalia Rocco , Picardo Francesco , Balsamo Teresa , Flammia Gerardo Paolo , Trombetta Domenico , Pantalone Angela , Kok Klaas , Ferronika Paranita , Muscarella Lucia Anna , Fazio Vito Michele
Published: July 2017

The Keap1/Nrf2 pathway is a master regulator of the cellular redox state through the induction of several antioxidant defense genes implicated in chemotherapeutic drugs resistance of tumor cells. An increasing body of evidence supports a key role for Keap1/Nrf2 pathway in kidney diseases and renal cell carcinoma, but data concerning the molecular basis and the clinical effect of its deregulation remain incomplete.

Here we performed a comprehensive genetic and epigenetic analysis of the KEAP1 gene in 37 tumor/normal paired tissues of clear cell Renal Carcinoma (ccRCCs). Promoter methylation analysis was performed by using a quantitative methylation specific PCR assay in real time, whereas mutation scanning was performed on FFPE tissues by direct sanger sequencing of the exons 4-7 codifying for the DGR domain of the Keap1 protein.

A tumor-specific DNA methylation of the KEAP1 gene promoter region was found in 18 out of 37 ccRCCs (48,6%) and a direct effects on the modulation of Keap1 mRNA levels was confirmed by in vitro 5-azacytidine treatment on three different ccRCCs cell lines. Analysis of an independent TGCA data set corroborate the epigenetic findings and reveals a significant correlation in multivariate analysis of epigenetic KEAP1 silencing with Overall Survival in ccRCCs.

Our results further suggest that epigenetic deregulation of the Nrf2/Keap1 system could play a pivotal role in the cancerogenesis of ccRCCs. In addition identifying patients with KEAP1 epigenetic abnormalities may contribute to disease progression prediction and response to therapy in ccRCC affected patients.

Citation Format: Federico Pio Fabrizio, Manuela Costantini, Massimiliano Copetti, Anna Maria la Torre, Angelo Sparaneo, Andrea Fontana, Maria Luana Poeta, Michele Gallucci, Steno Sentinelli, Paolo Graziano, Paola Parente, Vincenzo Pompeo, Laura De Salvo, Giuseppe Simone, Rocco Papalia, Francesco Picardo, Teresa Balsamo, Gerardo Paolo Flammia, Domenico Trombetta, Angela Pantalone, Klaas Kok, Paranita Ferronika, Lucia Anna Muscarella, Vito Michele Fazio. Epigenetic silencing in clear renal cell carcinoma: KEAP1 promoter hypermethylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2397. doi:10.1158/1538-7445.AM2017-2397
Abstract 2409: Loss of L1TD1 suppresses Notch1 induced LINE1 methylation and genomic stability
Authors: Chiu Chia-Chen , Huang Wei-Chen , Lee Kuan-Der , Chen Chih-Cheng , Hsu Chia-Chen , Kang Mei-Ling , Leu Yu-Wei , Hsiao Shu-Huei
Published: July 2017

The silencing of endogenous retrotransposon like LINE1 is critical for the maintenance of genomic stability and the LINE-1 type transposase domain containing 1 (L1TD1) gene possesses the repeated, putative LINE-1 RNA-binding domains and was hypothesized to regulate the activity of LINE-1 through DNA methylation. To verify if L1TD1 is responsible for LINE-1 silencing and the maintenance of somatic genome stability, CRSPR/Cas9 system was used to knockout (KO) L1TD1 locus in gastric, colon and breast cancer cells so as in the mesenchymal stem cells (MSCs). We observed that L1TD1 KO blocked the entrance of SUV39H1, histone methyl transferase, into cell nuclear which suppressed the tri-methylation of histone 3 at lysine 9 (H3k9me3) and LINE-1 methylation. A global demethylation and reduced HP1α recruitment were also observed. The global distribution of CTCF binding loci was also distorted as well as the bivalent histone marks. Further, L1TD1 KO distorted normal distribution of RassF1A expression, cytoskeleton conformation and therefore cell stiffness. The MSC-to-neuron differentiation was also blocked by the KO. Further, we found that overexpressed Notch-1 increased DNA methylation within LINE1 promoter, and this increase was attenuated by L1TD1 KO. Therefore, we conclude that the external signals like Notch1 affect L1NE1 methylation and genome stability through possible LINE1 interacting L1TD1. Since L1TD1 hypermethylation was observed in colon (n=100), gastric (n=19) and breast (n=79) cancers, L1TD1 abnormality is then a candidate for cancer biomarker. (Supported by: MOST-105-2320-B-194-004, MOST-105-2314-B-182A-134, MOST Taiwan and CMRPG6F0092, Chang Gung Memorial Hospital, Chia-Yi, Taiwan)

Citation Format: Chia-Chen Chiu, Wei-Chen Huang, Kuan-Der Lee, Chih-Cheng Chen, Chia-Chen Hsu, Mei-Ling Kang, Yu-Wei Leu, Shu-Huei Hsiao. Loss of L1TD1 suppresses Notch1 induced LINE1 methylation and genomic stability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2409. doi:10.1158/1538-7445.AM2017-2409
Abstract 2412: Monitoring small molecules and macromolecules methyltransferase activities using homogenous luminescent assay
Authors: Goueli Said A. , Hsiao Kevin
Published: July 2017

It is well recognized that methylation/demethylation of not only DNA, RNA, Proteins but also methylation of small molecules play major roles in modulation of the epigenome, transcriptome, neurotransmitter uptake and metabolic regulation. Recent biochemicval and biological data suggest that the activity of these enzymes and their expression level are under very strict regulation and any abnormal alteration in either one or both results in a wide varieties of pathogenic conditions such as cancer, inflammation, and neurodegenerative diseases. In addition to chromatin modulation, altered methylation of DNA and more recently mRNA have been recognized to regulate the transcriptome and the rate of message translation and stability. Furthermore, methylation of small molecules such as catechols and nicotinamide play critical roles in neurotransmitters uptake and function, and metabolic regulation, respectively. Thus, pharmacological modulation of these enzymes by small molecules will be beneficial in developing novel therapeutics for multiple unmet medical needs. Towards this goal of searching for activators/inhibitors of these enzymes for the development of next generation of drugs, screening assays for these modulators are urgently needed. To address these unmet needs, we have developed a novel assay that monitors the activities of these enzymes and their modulation by small molecules. The assay is bioluminescent based, HTS formatted and highly sensitive. A unique feature of this assay is its universality since it is based on monitoring the formation of the universal product S-adenosylhomocysteine (SAH), i.e., capable of detecting changes in activity of a broad range of methyltransferases such as DNA, RNA, protein, and small molecules. In addition, the assay has been validated for all classes of protein methyltransferases (Lysine and Arginine), and with different types of substrates (small peptides, large proteins, or even nucleosomes). This enables determining the specificity of these enzymes and their substrate requirements. The assay has high signal to background, low C.V., robust (Z’ value > 0.7), and has been validated using various plate densities such as 96-, 384, and 1536-well plates. A strong feature of this assay is its utility with broad range of substrates concentrations or the composition of the substrates (short vs. long peptides), thus enabling the generation of kinetic data and determining the mechanism of action of various modulators of methyltransferases of interest.

Citation Format: Said A. Goueli, Kevin Hsiao. Monitoring small molecules and macromolecules methyltransferase activities using homogenous luminescent assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2412. doi:10.1158/1538-7445.AM2017-2412
Abstract 2418: Characterization of the PAX8 regulatory network in epithelial ovarian cancer
Authors: Corona Rosario I. , Adler Emily , Lee Janet M. , Rodriguez-Malave Norma , Mhawech-Fauceglia Paulette , Sowter Heidi , Hazelett Dennis J. , Lawrenson Kate , Gather Simon A.
Published: July 2017

PAX8 is a lineage-restricted transcription factor expressed in a large proportion of epithelial ovarian cancers (EOCs). PAX8 is commonly upregulated in EOCs relative to precursor tissues, suggesting it functions as an oncogene. However, the biological role of PAX8 during cancer initiation and development is poorly understood, and the genome-wide transcriptional targets have yet to be comprehensively catalogued. Using stable models of PAX8 knockdown in HEYA8 and IGROV1 EOC cell lines we show that PAX8 knockdown reduces cell proliferation in vitro and tumor growth in vivo. To understand how PAX8 regulates neoplastic phenotypes in cancer cells we performed RNA expression profiling by microarray in HEYA8 and IGROV1 before and after PAX8 knockdown. We also performed chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) for PAX8 and a marker of active chromatin (H3K27ac) in both cell lines. De novo motif discovery in the ChIP-seq profiles identified a PAX-like binding motif and several potential PAX8 cofactors in IGROV1 that are not enriched in HEYA8. These cofactors include members of the TEAD and Sp/KLF families of transcription factors. We divided the genes from our differential expression (DE) analysis in PAX8 knockdown into three non-overlapping categories: (a) direct regulatory targets (RTs), i.e. DE genes with PAX8 binding sites in their promoter regions; (b) putative enhancer RTs, where DE genes and PAX8 binding sites reside within the same topological association domain (TAD) defined from embryonic stem cells; and (c) indirect RTs. Our findings reveal that PAX8 can function as either an activator or a repressor, and that PAX8 regulates a very different set of genes in each cell line, as only a small fraction RTs (~50) is shared between HEYA8 and IGROV1. Nonetheless, independent pathway enrichment analysis reveals four pathways downstream of PAX8 that are common to both cell lines: DNA replication, cellular response to lipopolysaccharides, TNFA signaling via NFKB and anatomical structure morphogenesis. Individually, we discovered enrichment for G2M checkpoint and EMT signaling in HEYA8, and tissue morphogenesis and mesenchymal cell development in IGROV1. Collectively, our results suggest that PAX8 regulates cell proliferation and cancer promoting processes via highly tissue-specific regulation of common oncogenic pathways.

Citation Format: Rosario I. Corona, Emily Adler, Janet M. Lee, Norma Rodriguez-Malave, Paulette Mhawech-Fauceglia, Heidi Sowter, Dennis J. Hazelett, Kate Lawrenson, Simon A. Gather. Characterization of the PAX8 regulatory network in epithelial ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2418. doi:10.1158/1538-7445.AM2017-2418
Abstract 2421: The role of p53 mutational status and SOX9 suppression in chemotherapy response of ovarian cancer
Authors: Cutter Noelle L. , Lucito Matthew , Frank Ryan , Doyle Kimberly
Published: July 2017

Ovarian cancers are highly heterogeneous where platinum based chemotherapy which induces DNA crosslinking resulting in apoptosis of the cell is the preferred treatment. However, many patients are intrinsically resistant or quickly develop resistance. The Sox factors are a large family of transcription factors that play important roles in tumor development and progression in a variety of human malignancies and diverse developmental processes, but their impact in clinical tumorigenesis is still unclear. An analysis of genomic changes in ovarian cancer has provided the most comprehensive and integrated view of cancer genes for any cancer type to date. Ovarian serous adenocarcinoma tumors from 500 patients were examined by The Cancer Genome Atlas (TCGA) Research Network and analyses are reported in a recent issue of Nature. This evidence suggests that epigenetic deregulation, such as methylation, may be a key factor in the onset and maintenance of chemoresistance. Previous microarray analysis results in our lab correctly identified a subset of about 300 genes that when methylated altered the chemoresistance of the ovarian epithelium cells in culture. Of the genes identified in the analysis we further set out to characterize oncogenes and tumor suppressor genes that interact with the guardian of the genome, TP53, to determine if we could elucidate the mechanism by which it increased resistance. Using several in-vitro assays, we determined that the loss of p53 in conjunction with SOX9 decreased the level of apoptosis in response to carboplatin. Furthermore, in cells with mutated p53/SOX9 show an increase in tumorigenesis. Regulation of several pathways with p53 mutations in ovarian cancer might represent a therapy response prediction and could be a future therapeutic target for ovarian cancer. In addition, the crosstalk between p53/SOX9 and epigenetic regulators may present a valid treatment option for increasing carboplatin sensitivity in resistant patients.

Note: This abstract was not presented at the meeting.

Citation Format: Noelle L. Cutter, Matthew Lucito, Ryan Frank, Kimberly Doyle. The role of p53 mutational status and SOX9 suppression in chemotherapy response of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2421. doi:10.1158/1538-7445.AM2017-2421
Abstract 2423: Bronchial airway gene expression signatures in mouse lung squamous cell carcinoma and their modulation by cancer chemopreventive agents
Authors: Xiong Donghai , Pan Jing , Zhang Qi , Szabo Eva , Miller Mark S. , Lubet Ronald A. , You Ming , Wang Yian
Published: July 2017

Due to exposure to environmental toxicants, a “field cancerization” effect occurs in the lung resulting in the development of a field of initiated but morphologically normal appearing cells in the damaged epithelium of bronchial airways with dysregulated gene expression patterns. Using a mouse model of lung squamous cell carcinoma (SCC), we performed transcriptome sequencing (RNA-Seq) to profile bronchial airway gene expression and found activation of the PI3k and Myc signaling networks in cytologically normal bronchial airway epithelial cells of mice with preneopastic lung SCC lesions, which was reversed by treatment with the PI3K Inhibitor XL-147 and pioglitazone, respectively. Activated MYC signaling was also present in premalignant and tumor tissues from human lung SCC patients. In addition, we identified a key microRNA, mmu-miR-449c-5p, whose suppression significantly up-regulated Myc expression in the normal bronchial airway epithelial cells of mice with early stage SCC lesions. We developed a novel bronchial genomic classifier in mice and validated it in humans. In the classifier, Ppbp (pro-platelet basic protein) was overexpressed 115 fold in the bronchial airways of mice with preneoplastic lung SCC lesions. This is the first report that demonstrates Ppbp as a novel biomarker in the bronchial airway for lung cancer diagnosis.

Citation Format: Donghai Xiong, Jing Pan, Qi Zhang, Eva Szabo, Mark S. Miller, Ronald A. Lubet, Ming You, Yian Wang. Bronchial airway gene expression signatures in mouse lung squamous cell carcinoma and their modulation by cancer chemopreventive agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2423. doi:10.1158/1538-7445.AM2017-2423
Abstract 2427: Alterations in gene expression after exposure of MDA-MB-231 cells to isobutylparaben
Authors: Strelchuk Christine , Taggart Holly Jones
Published: July 2017

Parabens are a group of chemical compounds extensively used as preservatives in consumer products such as cosmetics, pharmaceuticals and processed food. Parabens have been shown to have endocrine disrupting properties, to accumulate in breast tissue and to increase the proliferation of hormone receptor positive breast cancer cell lines through competitive binding to the estrogen receptor. Studies have shown that isobutylparaben has the highest binding affinity for the aryl hydrocarbon receptor (AhR), reported to bind environmental toxins. This study examined the alterations in gene expression correlated to exposure of the hormone receptor negative breast cancer cell line MDA-MB-231 to isobutylparaben over a 72 hour time frame. Exposure of the cells to concentrations of isobutylparabens commonly found in consumer products alters the genetic expression profile of the cells. In our study, detectable expression levels of AhR mRNA decreased, while cytochrome P450 (CYP1A1) and receptor activator of NFκB ligand (RANKL) mRNA levels increased, as analyzed by qPCR. The downregulation of AhR and altered expression levels of invasion related genes is suggestive of an increased metastatic phenotype with exposure to isobutylparaben.

Citation Format: Christine Strelchuk, Holly Jones Taggart. Alterations in gene expression after exposure of MDA-MB-231 cells to isobutylparaben [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2427. doi:10.1158/1538-7445.AM2017-2427
Abstract 2428: The cytidine deaminase APOBEC3B is a regulator of gene expression in breast cancer
Authors: Periyasamy Manikandan
Published: July 2017

The APOBEC3B (A3B) cytidine deaminase gene has been implicated driving the mutational landscape in breast and other cancer types. We have discovered that A3B is a co-activator of estrogen receptor (ER) mediated gene expression in breast cancer. Mechanistic studies established that A3B regulates gene expression by promoting C-to-U deamination at ER binding regions. We show that these C-to-U changes lead to the generation of DNA strand breaks through activation of base excision repair (BER) and to repair by non-homologous end-joining (NHEJ) pathways. We have performed RNA-seq and A3B ChIP-seq, in multiple ER+ and ER- breast cancer cell lines. Remarkably, these studies demonstrate that A3B regulates the expression of many genes in inflammatory and interferon signaling pathways, in addition to the regulation of ER target genes. These findings, together with other data to be presented, provide a clear role for A3B in transcription regulation. These findings also raise the possibility that transcription factor mediated recruitment of A3B to DNA may facilitate acquisition of A3B driven cancer mutations.

Citation Format: Manikandan Periyasamy. The cytidine deaminase APOBEC3B is a regulator of gene expression in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2428. doi:10.1158/1538-7445.AM2017-2428
Abstract 2429: Generation of a transcriptome of oncogenic mutant p53 in triple-negative breast cancer
Authors: Steele Rebecca E. , McDade Simon S. , Mullan Paul B.
Published: July 2017

The purpose of this study was to generate a transcriptional profile specific for mutant p53 in triple-negative breast cancer (TNBC), representing potential novel therapeutic targets. The highly heterogeneous TNBC tumor subgroup is established as having a poor clinical outcome. There are currently no targeted therapies for women with TNBC; consequently, there is an unmet clinical need to identify targetable pathways. Approximately 75% of these tumors are known to harbour a mutation in Tp53 (mutp53). Chromatin Immunoprecipitation Sequencing (ChIP-Seq) for mutp53 and ETS-1 (a known mutp53 interactor) was carried out using the D0-1 (sc-126) and sc-20 antibodies respectively, in the MDA-MB-468 TNBC cell line. The aim of these ChIP-seq experiments was to identify sites bound by endogenous R273H mutp53 and ETS-1 and to confirm that target genes were co-regulated. We identified 282 novel mutp53 specific genomic binding sites and 37 mutant p53 and ETS1 shared binding sites. In-house data analysis and peak calling generated a list of potential transcriptionally co-regulated genes adjacent to these genomic sites. PDGFβ, TRIB3, BTRC, TNFSF18 & PI3 were successfully validated as being transcriptionally regulated by R273H mutp53 and not wild-type p53 (as shown by MCF7 ChIP-seq comparisons). A number of these genes are known to be aberrantly expressed in TNBC patient tumors and harbor interesting immune biology, providing potential opportunities for novel therapeutic strategies. In addition, DNA damaging chemotherapy-resistant MDA-MB-468 derivatives express elevated levels of mutp53 protein and altered expression of our validated transcriptional targets, suggesting that they may also have roles in promoting resistance to current chemotherapy treatments, downstream of mutp53. In conclusion, we have identified a cohort of ‘mutp53 specific’ transcriptional targets which may have roles in modulating immune responses to TNBCs, driving resistance to current chemotherapies and could provide opportunities for the development of novel treatments for TNBCs.

Citation Format: Rebecca E. Steele, Simon S. McDade, Paul B. Mullan. Generation of a transcriptome of oncogenic mutant p53 in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2429. doi:10.1158/1538-7445.AM2017-2429
Abstract 2436: Non canonical activation of Sonic Hedgehog pathway in uterine leiomyosarcoma
Authors: Garcia Natalia , Oliveira Bianca C. , Ferreira Kelly P. , Baracat Edmund C. , Carvalho Katia C.
Published: July 2017

Background: Uterine leiomyoma (ULM) and leiomyosarcoma (ULMS) are smooth muscle tumors with distinct clinical and biological behavior. They can be a cause of infertility and even death. Little is known about the factors that can to influence these tumors behavior and biology. Sonic Hedgehog (SHH) pathway components were previously implicated in the ULMS malignancy risk; however its activation mechanism is unknown. In this study, we investigated the gene expression profile of Sonic Hedgehog Signaling and pathway upstream genes in myometrium (MM), ULM and LMS cell lines.

Methods: Total RNA obtained from cell lines were submitted to cDNA synthesis and quantitative real time PCR (qRT-PCR), using 7500 System (Life Technologies, USA). Gene expression of SHH, PTCH1, SMO, SUFU, GLI 1-3, CCND1, BCL-2 and BMP4 were assessed.

Results: Gene expression data showed an upregulation of SMO, SUFU, GLI1, BCL-2 and BMP4 in LMS cells when compared to MM. GLI1 (RQ=11) and BCL-2 (RQ=20) genes had higher expression. In the other side, SHH, PTCH1, GLI2 and GLI3 showed upregulation in MM cells.

Conclusion: Our results showed that the Sonic hedgehog pathway is activated in LMS cells but by SHH independent form (non-canonical pathway). This could be explained by the downexpression of SHH ligand and the PTCH1 receptor, and by the upexpression of SMO, GLI1 and pathway target genes as BCL-2 and BMP4 in uterine LMS cells compared to LM and MM. These results suggest a new therapeutic perspective for LMS by target drugs to inhibit SMO and GLI1 molecules.

Citation Format: Natalia Garcia, Bianca C. Oliveira, Kelly P. Ferreira, Edmund C. Baracat, Katia C. Carvalho. Non canonical activation of Sonic Hedgehog pathway in uterine leiomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2436. doi:10.1158/1538-7445.AM2017-2436
Abstract 2437: Overexpression of MYL5 promotes cervical cancer cell metastasis through SLUG and HIF-1α signaling
Authors: Zhang Lan
Published: July 2017

Myosin light chains (MLC) serve important regulatory functions in a wide range of cellular and physiological processes. Previous research found that MLC are also chromatin-associated nuclear proteins which regulate gene transcription. In this study, we show that the MLC member myosin regulatory light chain 5 (MYL5) controls metastasis in cervical cancer. We uncover this role of MYL5 through clinical cervical cancer samples and various in vitro and in vivo models of metastasis. MYL5 promotes metastasis by enhancing the transcription of SLUG and hypoxia inducible factor-1α (HIF-1α). Moreover, we reveal a bidirectional regulation between MYL5 and HIF-1α. HIF-1α specifically binds to the MYL5 promoter region, and hypoxia leads to increased levels of MYL5 in cervical cancers. Overexpression of MYL5 sustained HIF-1α expression in normoxic condition. Clinical data confirmed a positive correlation between MYL5 and HIF-1α. In summary, our observations suggest a potential application of MYL5 in prognosis prediction and cancer treatment.

Citation Format: Lan Zhang. Overexpression of MYL5 promotes cervical cancer cell metastasis through SLUG and HIF-1α signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2437. doi:10.1158/1538-7445.AM2017-2437
Abstract 2440: Cross-tissue interactome analyses unravel novel oncogenic roles of SOX2
Authors: Schaefer Thorsten , Candido Silvia , Wang Hui , Bock Thomas , Schmidt Alexander , Lengerke Claudia
Published: July 2017

First recognized for its significance in early embryogenesis, SOX2 (sex determining region Y box 2) more recently received major attention as a (i) transcriptional master regulator of stemness driving the re-programming of terminally differentiated somatic cells back into a pluripotent stem cell state and (ii) powerful oncogene of the cancer stem cell (CSC) compartment. We and others have shown that SOX2 expression relies on canonical PI3K/AKT signaling involving direct physical contact and phospho-modification by AKT. Interestingly, we observed that these interactions are highly tissue specific which prompted us to perform a systematic proteomic analysis of co-factors regulating SOX2 biology.

The SOX2 interactome was analyzed in human breast carcinoma and glioblastoma cell lines by co-immunoprecipitation (co-IP) and HPLC/MS revealing >900 individual and >70 conserved proteinaceous co-factors, thus underscoring a hitherto underestimated scaffolding function of SOX2. As expected, a major proportion of binding partners cluster in functional categories implicated in DNA binding and/or nucleotide modification (e.g. helicases, transferases, and ligases). However, a near equal share of SOX2 associated factors fall into functional classes formally unrelated to transcription modulation such as cell adhesion, cytoskeletal organization, or metabolic adaptation. Using SOX2 knock-down and overexpression cells generated by lentiviral technology, we confirm co-regulation of SOX2 with selected binding partners. Moreover, we observe co-depletion of SOX2 and protein co-factors in cells of stalled AKT activity, and a molecular restoration of both upon proteasomal inhibition.

We conclude that, besides its known significance in DNA activity control, the SOX2 protein engages in various interactions seemingly unrelated to transcription modulation. These newly discovered interactions are likely to synergize with canonical SOX2 functions (i.e. the induction and maintenance of stemness), but involve regulation at post-translational level and of extra-nuclear components. Our efforts thus add an exciting new facet to SOX2 biology and eventually, may open new therapeutic avenues for targeted anti-CSC therapy.

Citation Format: Thorsten Schaefer, Silvia Candido, Hui Wang, Thomas Bock, Alexander Schmidt, Claudia Lengerke. Cross-tissue interactome analyses unravel novel oncogenic roles of SOX2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2440. doi:10.1158/1538-7445.AM2017-2440
Abstract 246: Assessing microbial dysbiosis of electronic cigarettes and cigarette smokers using oral and lung microbiome
Authors: Ying Kevin , Song Min-Ae , Weng Daniel Y. , Nickerson Quentin , Frankhouser David , Yan Pearlly S. , Bundschuh Ralf , Wewers Mark D. , Mathé Ewy , Freudenheim Jo L. , Shields Peter G.
Published: July 2017

The link between smoking tobacco and changes in the oral microbiome in response to tobacco smoking are well established. It is not known if there are changes in response to electronic cigarettes (e-cig). These changes in the microbiome are associated with increased numbers of disease causing pathogens. Currently there are no published studies that have investigated the relationship of smoking tobacco on both the oral and lung microbiome. There is insufficient evidence showing whether changes in oral cavity and lung microbiome are also seen in e-cig users. We will study the oral cavity and lung of non-smokers, smokers and e-cig users to examine concordance between oral cavity and the lungs as well as comparing the three groups, examining the microbiomes and expression of inflammatory markers. We hypothesize that microbial dysbiosis and expression of inflammatory cytokines will differ for smokers and non-smokers; and that e-cig users will have microbial dysbiosis similar to cigarette smokers. A cross-sectional study is being conducted on three groups, 1) never-smokers, 2) cigarette smokers, and 3) e-cig users. For each study participant, saliva and bronchoalveolar lavage (BAL) are being collected to measure microbiome. RNA is extracted from saliva and BAL samples for total transcriptome analysis using RNA-seq. This analysis will detect human and bacterial reads thereby allowing observations of bacterial communities as well as human inflammatory cytokine response to bacterial presence. 85% to 98% of BAL sample reads aligned to the human genome compared to less than 50% from saliva samples. The alignment results allow us to deduce that the majority of reads from BAL samples are human and that the majority of the reads in saliva samples are bacterial.

Preliminary results show detection of human RNA expression and of bacterial reads are present in both saliva and BAL samples. More samples are being processed and the comparison of BAL and saliva samples between the three groups will be discussed.

Citation Format: Kevin Ying, Min-Ae Song, Daniel Y. Weng, Quentin Nickerson, David Frankhouser, Pearlly S. Yan, Ralf Bundschuh, Mark D. Wewers, Ewy Mathé, Jo L. Freudenheim, Peter G. Shields. Assessing microbial dysbiosis of electronic cigarettes and cigarette smokers using oral and lung microbiome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 246. doi:10.1158/1538-7445.AM2017-246
Abstract 2460: Copy number alterations in NOTCH2 and PTP4A3 are associated with prognosis and support novel therapeutic strategies for malignant pleural mesothelioma
Authors: Leon Leticia G. , Gemelli Maria , Zucali Paolo A. , Zoumadakis Christos , Giovannetti Elisa
Published: July 2017

Malignant pleural mesothelioma(MPM) is an aggressive cancer, which incidence has constantly increased over the past two decades and is expected to peak in 2020. The overall prognosis is poor, and predictive biomarkers of drug activity are missing. Given the heterogeneous and complex nature of MPM, it is likely that genomic aberrations changing the expression of several genes, might affect therapeutic response. Therefore, the aim of the present study was to identify genes whose copy number alterations might predict the MPM prognosis.

Recurrent copy number alterations of genes were analyzed by high-resolution whole-genome sequencing in DNA obtained from a “discovery cohort” of 26 resected MPM patients treated with pemetrexed-based chemotherapy (8 with progressive disease, vs. 10 with stable disease and 8 with partial response). Prognostic markers identified by Copy Number Variation analysis with Nexus, Control-FREEC and ReadDepth software were validated by PCR gene copy number and gene expression analyses both in the “discovery” and in two “validation” cohorts of pemetrexed-treated and untreated patients (N=45 and 40). The role of emerging genes was evaluated through siRNA and pharmacological studies using proliferation, migration and apoptosis assays in MPM cells.

As reported previously we observed copy number loss of CDKN2A (15.4%) and BAP1 (7.7%). Interestingly, copy number gain of NOTCH2 was observed in 50% of samples of the patients who underwent progression, whereas losses of PTP4A3 were associated with clinical benefit (SD+PR). The prognostic relevance of NOTCH2 was confirmed by PCR analysis. NOTCH-2 silencing reduced MPM cell migration and enhanced apoptosis induction by pemetrexed, while a PTP4A3 inhibitor overcame pemetrexed resistance in MPM cells characterized by high NOTCH2 and PTP4A3 expression.

These results support the role of NOTCH2 as a novel prognostic/predictive biomarker for MPM, prompting prospective randomized trials for its validation. Moreover, preclinical data suggest that NOTCH2 and PTP4A3 are oncogenes suitable for effective therapeutic targeting in pemetrexed-resistant MPM cells.

Citation Format: Leticia G. Leon, Maria Gemelli, Paolo A. Zucali, Christos Zoumadakis, Elisa Giovannetti. Copy number alterations in NOTCH2 and PTP4A3 are associated with prognosis and support novel therapeutic strategies for malignant pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2460. doi:10.1158/1538-7445.AM2017-2460
Abstract 2464: Papillary renal cell carcinoma, proposal of a new classification system based on integrated molecular, histological and clinical analysis
Authors: Saleeb Rola , Farag Mina , Brimo Fadi , Rotondo Fabio , Plant Pamela , Yousef George
Published: July 2017

Background:

Papillary Renal Cell Carcinoma (PRCC) is divided into histological subtypes 1 and 2. Type 2 is known to have worse clinical behavior. A number of PRCC cases (~ 50%), fail to meet all reported morphological criteria for either type, hence are best characterized as PRCC not otherwise specified (NOS). There are yet no reliable markers to resolve the PRCC NOS category. That in turn reflects the clinical dilemma of how to manage these patients.

Experimental Design:

PRCC patient cohort of 115 cases was selected for the study. Cases were subtyped histologically into PRCC types 1, 2 and NOS. Potentially distinguishing markers ABCC2, CA9, SAll4, and BCL2 selected from our previous genomic analysis, were assessed by immunohistochemistry (IHC). A total of 24 cases were further selected for molecular analysis using miRNA expression and copy number variation (CNV). Univariate and multivariate survival analysis were performed using Log rank test and cox proportionate hazards.

Results:

Markers ABCC2, CA9 exhibited distinct staining patterns between the two classic PRCC subtypes; and successfully classified many of the PRCC NOS (45%) cases. Moreover, immunomarkers revealed a third distinct subtype of PRCC (35% of the PRCC cohort). Molecular

testing using miRNA expression and CNV analysis confirmed the presence of three distinct molecular signatures corresponding to the 3 subtypes. On univariate analysis DFS was significantly enhanced in the type1 versus 2& 3 (p value 0.047). PRCC subtyping retained

significance on multivariate analysis (p value 0.025, HR:6, 95% CI 1.25 to 32.2) .

Conclusion:

We propose a new classification system of PRCC integrating morphological, immunophenotypical, and molecular analysis. Our classification reveals a 3rd PRCC subtype that was not previously described. This subtype has overlapping morphology of with PRCC types 1 and 2, hence would be subtyped as PRCC NOS in the current classification. Molecularly PRCC type 3 has a distinct signature and clinically it behaves similar to PRCC type 2. The new classification stratifies PRCC patients into clinically relevant subgroups and has significant future implications on the management of PRCC.

Citation Format: Rola Saleeb, Mina Farag, Fadi Brimo, Fabio Rotondo, Pamela Plant, George Yousef. Papillary renal cell carcinoma, proposal of a new classification system based on integrated molecular, histological and clinical analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2464. doi:10.1158/1538-7445.AM2017-2464
Abstract 2471: Role of PALB2-BRCA1 interaction in tumor suppression
Authors: Mahdi Amar H. , Huo YanYing , Xia Bing
Published: July 2017

Homologous recombination (HR) is the only error-free pathway for the repair of DNA double strand breaks (DSBs). BRCA1 and BRCA2, the two major breast cancer suppressor proteins, play essential roles in HR-mediated repair of DSBs by promoting the recruitment of RAD51, the recombination enzyme, to DNA damage sites for the initiation of HR. PALB2 (partner and localizer of BRCA2) plays a key role in this pathway by acting as a chromatin adaptor for BRCA2 and a linker between BRCA1 and BRCA2. Like BRCA1 and BRCA2, PALB2 is a tumor suppressor gene itself. Germline, heterozygous mutations in the gene increase the risk of breast, ovarian and pancreatic cancers. However, its mechanism is not fully understood. To investigate the in vivo role of the PALB2-BRCA1 interaction, we previously generated a Palb2 knockin mouse strain which contains a mutation that disrupts BRCA1 binding. This mouse model also allows us to bypass the embryonic lethality of the Palb2 KO mice. In this study, we hypothesized that the direct communication between the two proteins is critical for proper DNA damage repair and response in vivo and for suppression of tumorigenesis. Indeed, both immunohistochemistry (IHC) and immunofluorescence (IF) demonstrated that different tissues of the mutant mice have higher levels of endogenous DSBs (γH2AX foci) and slower DSB repair kinetics after ionizing radiation (IR). Yet, mutant cells were more resistant to cell death. When aged under normal conditions, mutant mice showed increased tumor incidence in multiple tissues, particularly in the liver. When challenged by IR, mutant mice quickly developed thymic lymphoma and later in other tissues including liver and ovary, etc. Interestingly, when crossed with Trp53 mutant mice, the resulting Palb2m/m;Trp53+/- mice showed greatly accelerated development of thymic lymphoma and osteosarcoma, which are typically associated with Trp53 but not Palb2 mutations. Exome sequencing revealed focal deletion of the wild-type allele of Trp53 in the majority of the tumors, suggesting that disruption of BRCA1-PALB2/BRCA2 axis promotes regional genomic deletions that may lead to loss of other tumor suppressors such as p53. Our results underscore the importance of the BRCA1-PALB2/BRCA2 pathway for tumor suppression and suggest a potentially novel mechanism for BRCA1/PALB2-mediated tumor suppression, which is by preventing Trp53/TP53 loss of heterozygosity (LOH), which allows for tumor development.

Citation Format: Amar H. Mahdi, YanYing Huo, Bing Xia. Role of PALB2-BRCA1 interaction in tumor suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2471. doi:10.1158/1538-7445.AM2017-2471