bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 729: Novel role of CB2R in tuning breast tumor microenvironment, EGF/EGFR and IGF-I/IGF-IR pathways
Authors: Elbaz Mohamad , Nasser Mohd , Ravi Janani , Ahirwar Dinesh , Ganju Ramesh
Published: July 2016

Cannabinoid receptor-2 (CB2R) is an integral part of the endocannabinoid system. It is upregulated in the primary breast cancer lesions and in different types of immune cells however; its functional role in breast tumorigenesis is not well understood. The present study was aimed at evaluating the mechanistic anti-tumor role of CB2R activation on breast cancer cells and immune cells within the breast tumor microenvironment. First, we analyzed the anti-tumorigenic mechanisms of CB2R activation in ERá- and ERá+ breast cancer cells. Our studies showed that CB2R specific agonist (JWH-015) inhibits EGF and IGF-I-induced migration and invasion of ERá+ and ERá- breast cancer cells. At the molecular level, JWH-015 inhibits EGFR and IGF-IR activation and their downstream targets STAT3, AKT, ERK, NF-kB and MMP-9/MMP-2. Interestingly, We found that JWH-015 significantly reduces breast cancer growth in vivo and the tumors that were derived from CB2R agonist treatment showed reduced activation of EGFR and IGF-IR and their downstream targets compared to control group. Since CB2R is overexpressed in immune cells, we assessed for the role of CB2R activation on modulation of immune cells present in tumor stroma. We observed increased tumor weight, more myeloid derived suppressor cells (MDSCs) (CD11b+/Gr-1+) and less CD3+/CD8+ cells in orthotopically injected CB2R knock out mice compared to wild type mice. Furthermore, we found that JWH-015-treated wild type mice have reduced tumor growth and metastasis, more CD3+/CD8+ cells and less MDSCs within the tumor stroma. For the first time, we show that CB2R activation might suppress breast tumor growth and metastasis through novel mechanisms of inhibiting EGFR and IGF-IR signaling axes on tumor cells and modulating the immune cells’ compositions within the breast tumor microenvironment.

Citation Format: Mohamad Elbaz, Mohd Nasser, Janani Ravi, Dinesh Ahirwar, Ramesh Ganju. Novel role of CB2R in tuning breast tumor microenvironment, EGF/EGFR and IGF-I/IGF-IR pathways. [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 729.
Abstract 731: Transcriptional control of inflammatory signaling in the megakaryocyte and erythroid lineages: implications for the tumor microenvironment
Authors: Trinh Bon Q. , Barengo Nicolas , Kim Sang Bae , Lee Ju-Seog , Zweidler-McKay Patrick A. , Naora Honami
Published: July 2016

Thrombocytosis, characterized by high platelet counts, and anemia, caused by low numbers of red blood cells, are associated with poor outcomes in various solid tumors and also occur in several myeloid disorders. The megakaryocyte and erythroid lineages give rise to platelets and red blood cells, respectively, but the mechanisms that cause increased megakaryopoiesis and concurrently decreased erythropoiesis in malignancies are poorly understood. We and others have previously identified that DLX4, a developmental transcription factor encoded by a homeobox gene, is aberrantly expressed in several types of solid tumors and myeloid malignancies but the mechanisms of DLX4 are poorly understood. In this study, we identified that DLX4 expression increases during megakaryopoiesis and decreases during erythropoiesis. Overexpression and knockdown experiments using normal CD34+ stem/progenitor cells and bipotent K562 cells demonstrated that DLX4 induces lineage markers and morphologic features of megakaryocytes, and concomitantly represses erythroid marker expression and hemoglobin levels. Gene Ontology and Gene Set Enrichment Analyses of genome-wide changes in gene expression induced by DLX4 showed that DLX4 induces a megakaryocytic transcriptional program and inhibits an erythroid transcriptional program. Bioinformatic analysis also revealed that DLX4 induces gene signatures that are associated with NF-kappaB signaling. The ability of DLX4 to promote megakaryocyte development and to inhibit erythroid differentiation was diminished by blocking NF-kappaB activity or by repressing IL1B, a transcriptional target of DLX4. These findings indicate that DLX4 promotes megakaryocyte development at the expense of erythroid generation in part by inducing inflammatory signaling. Studies are ongoing to investigate the transcriptional control by homeobox genes of inflammatory signaling in thrombocytosis and anemia that are associated with solid tumors and myeloid disorders.

Citation Format: Bon Q. Trinh, Nicolas Barengo, Sang Bae Kim, Ju-Seog Lee, Patrick A. Zweidler-McKay, Honami Naora. Transcriptional control of inflammatory signaling in the megakaryocyte and erythroid lineages: implications for the tumor microenvironment. [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 731.
Abstract 751: Secreted anterior gradient-2 is a potential tumor-microenvironment-related therapeutic target
Authors: Guo Hao , Zhu Qi , Wu Zhenghua , Mangukiya Hitesh B. , Yu Xiaoyan , Li Dawei
Published: July 2016

The secreted anterior gradient-2 (AGR2) orthologs in amphibians are critical signaling proteins in anterior specification and limb regeneration. It is also overexpressed in diverse human cancers especially adenocarcinomas. Although tumor-related functions of intracellular AGR2 have been intensively investigated and several AGR2-related signaling pathways have been identified, the full scope of secreted AGR2 functions, especially the exact mechanism of actions, remain insufficiently explored. Here we report that AGR2 induction after SKOV3 xenograft inoculation is tumor microenvironment (TME) dependent. This phenomenon can be mimic in vitro through serum deprivation but not hypoxia treatment. In addition, the secreted AGR2 may in turn influence the development of TME. It scatters the tumor cells and guides the nearby vascular endothelial cells and fibroblasts toward the source of AGR2 through various degrees of crosstalk with VEGF and bFGF pathways. Blocking the AGR2 inhibits the tumor growth and decreases the blood vessel density in SKOV3 xenograft model. Our results show that exogenous AGR2 binds specifically with VEGF and bFGF, and promotes their dimerization. This binding enhances the phosphorylation signals, such as ERK phosphorylation, induced by VEGF and bFGF, and promotes the VEGF- and bFGF- dependent angiogenesis. We also find that heparin may reverse the binding between bFGF and AGR2, indicating that dimerized bFGF that promoted by AGR2 would be released when it is close to the cell surface where heparin sulfate is abundant. Taken together, the present results suggest that tumor-secreted AGR2 may activate the specific growth factors surrounding the tumor cells to promote the angiogenesis and tumor growth, and also act as a short-range chemoattractant to guide surrounding vascular endothelial cells and fibroblasts toward tumor cells in building the TME. This mechanism of action directly links the secreted AGR2 with the cell signaling networks well-known in building TME. This may help to further explain how tumor cells interact with and attract nearby fibroblasts and blood vessel cells to form their microenvironment and support their growth, suggesting a potential anti-TME target.

Citation Format: Hao Guo, Qi Zhu, Zhenghua Wu, Hitesh B. Mangukiya, Xiaoyan Yu, Dawei Li. Secreted anterior gradient-2 is a potential tumor-microenvironment-related therapeutic target. [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 751.
Abstract 755: ￼Antagonistic effect of M-MDSC and N-MDSC during astrocytoma progression
Authors: Huang. Chin-NIng
Published: July 2016

The traditional dogma holds that tumor size has a positive correlation with tumor progression. However, on murine orthotopical astrocytoma model ALTS1C1, we found that the tumor growth is not a linear function instead a shrinkage and regrowth wave was noticed. To examine the un-characteristic tumor growth behavior, the relative relationship among tumor cells, brain resident cells, such as astrocytes and microglia, and infiltrating cells, such as monocytic (CD45+CD11b+) or lymphocytic (CD45+CD11b-) lineage cells, was analyzed by flow cytometry and immunohistochemistry (IHC). Not only tumor area, peripheral organs, including spleen, blood, and lymph nodes were also examined. The preliminary data showed that in tumor shrinkage stage, CD3+ lymphocytes increased dramatically, while CD11b+CD45low microglia decreased. Furthermore, we found that the change of two subtypes of myeloid derived suppressor cells (MDSCs), Ly6G+ neutrophilic MDSC (N-MDSC) and Ly6G- monocytic MDSC (M-MDSC), was in opposite direction during tumor progression. The increase of M-MDSCs is associated with the increase of tumor size while the N-MDSC is decreased. More interestingly, the flow cytometry data shows that the total amount of CD11b+ macrophages was constant during tumor growth phase, but IHC shows the different distribution pattern of macrophage subsets within different tumor regions (invasion tumor front vs tumor core). Currently, we are examining the dynamic change of these cells following irradiation to identify the cells that are responsive for tumor shrinkage and re-growth following radiation therapy for brain tumor. These results will be presented in the meeting.

Citation Format: Chin-NIng Huang. ￼Antagonistic effect of M-MDSC and N-MDSC during astrocytoma progression. [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 755.
Abstract 765: The manipulation of FAP expression and its role in SDF-1 and IL-6 secretion by fibroblasts and melanoma cells
Authors: Fisher Zachary B. , Miyamoto Yuko J.
Published: July 2016

The cancer microenvironment surrounds tumors and primarily consists of fibroblasts and immune cells that cancer cells utiliaze to support their growth and invasion. Cancer-associated fibroblasts (CAFs) are the most prominent cell type in the microenvironment. They are attracted to tumors and activated by signals from cancer cells, including transforming growth factor-â (TGF-â). Following activation, CAFs display FAP on their surface, which breaks bonds in the extracellular matrix (ECM), creating space for cancer cells to migrate. CAFs also secrete cytokines, including stromal cell-derived factor-1 (SDF-1) and interleukin-6 (IL-6), to initiate this migration. This study seeks to determine if there is a link between the expression of fibroblast activation protein (FAP) and the specific production of SDF-1 and IL-6. Flow cytometry was used to verify the expression of FAP by the Malme-3 fibroblast cell line at an average rate of 50.6% and in the Malme-3M melanoma cell line at 4.8%. TGF-â treatment increased FAP expression in Malme-3M melanoma cells (50% increase), indicating a prominent role in cell transformation. siRNA transfection was also used to reduce FAP expression in both the fibroblasts (48.4% decrease) and the melanoma cells after initial TGF-â activation (11.6% decrease). Preliminary data from intracellular staining and ELISA analysis suggest that SDF-1 and IL-6 production both increased in melanoma cells in direct correlation to FAP expression in, but may be inversely related in fibroblasts. Future studies will further analyze the FAP expression levels and cytokine secretion of both cell types in different microenvironments to elucidate the relationship between FAP expression in cancer and the secretion of SDF-1 and IL-6.

Citation Format: Zachary B. Fisher, Yuko J. Miyamoto. The manipulation of FAP expression and its role in SDF-1 and IL-6 secretion by fibroblasts and melanoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 765.
Abstract 791: Examining the roles of deleterious and potentially damaging variant alleles in African and Caucasian ancestral populations
Authors: Gibert Myron K. , Ricks-Santi Luisel , McDonald John T.
Published: July 2016

The purpose of this study was to analyze the overlap between potentially damaging Single Nucleotide Polymorphisms (SNPs) for coding gene regions across African American and Caucasian populations. Allele frequency and genotype data from the International HapMap Project was acquired for ancestral groups from Utah Residents with Northern and Western European Ancestry (CEU), Yoruba in Ibadan, Nigeria (YRI) and African Americans from the Southwest (ASW) resulting in 34,844 unique variants. Using the Variant Effect Predictor, non-synonymous SNPs in overlapping transcripts were filtered and found in 12,367 unique genes. SIFT or Polyphen-2 scores were used to determine which variants were likely to prove damaging to protein function, resulting in 10,917 variants. Functional annotation of biological pathways in genes affected by these variants was analyzed using DAVID. There was enrichment in olfactory pathways/g-protein coupled receptor function, cell membrane function and epidermal growth factor activity. Comparisons of genes affected by these deleterious variants were overlapped with the catalog of published genome-wide association studies (GWAS) with SNP-traits associated with cancer. There were 90 of 463 genes in common with this data that were examined for linkage disequilibrium with the damaging variants. Finally, SNPs with a variant allele frequency differences >±5% between CEU versus YRI or ASW resulted in with 2,381 SNPs with 55.7% overlap between groups. Analysis of patient genotypes found those in the CEU population were more likely to possess a homozygous allele. Using Chi-Squared tests, populations were examined for risk allele frequencies by genotype. This study demonstrated variants with potentially damaging consequences in cancer pathways are disproportionally represented in different ancestral populations.

Citation Format: Myron K. Gibert, Luisel Ricks-Santi, John T. McDonald. Examining the roles of deleterious and potentially damaging variant alleles in African and Caucasian ancestral populations. [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 791.
Abstract 805: The molecular mechanisms of obesity driving breast cancer etiology and prognosis in post-menopausal women
Authors: Heng Yu Jing Jan , Wang Jun , Hazra Aditi , Hunter David J. , Eliassen A. Heather , Tamimi Rulla M. , Hankinson Susan E. , Beck Andrew H.
Published: July 2016

This study aims to gain insights into the molecular mechanisms of obesity driving breast cancer etiology and prognosis as post-menopausal women with high body mass index (BMI) have increased breast cancer risk and poorer prognosis.

We examined the differential gene expression of breast tumors from 441 post-menopausal women part of the Nurses’ Health Studies. Primary analysis was performed between women with BMI of <25, ≥25 to <30, ≥30 to 35 and ≥35 while secondary analysis was performed using BMI as a continuous variable. Analyses were adjusted for age of diagnosis, year of diagnosis, post-menopausal hormone therapy, alcohol consumption, microarray batch and ER status.

In the primary analysis, differentially expressed genes associated with BMI were identified in pairwise analyses comparing women with BMI ≥35 with women in lower BMI categories (<25 vs. ≥35, n = 58 significant genes; ≥25 to <30 vs. ≥35, n = 29; ≥30 to 35 vs. ≥35, n = 12; FDR<0.05); no significant genes were identified in the other pairwise comparisons. In the secondary analysis which considered BMI as a continuous value, two genes, NCOA3 and PTPN1, were significantly up-regulated in breast tumors with increasing BMI (FDR<0.05). Gene set enrichment analyses suggested that tumors from post-menopausal women with increasing BMI have increased inflammation and decreased carcinogen/anti-cancer drug metabolism.

These molecular insights have further elucidated breast cancer etiology in post-menopausal women with high BMI.

Citation Format: Yu Jing Jan Heng, Jun Wang, Aditi Hazra, David J. Hunter, A. Heather Eliassen, Rulla M. Tamimi, Susan E. Hankinson, Andrew H. Beck. The molecular mechanisms of obesity driving breast cancer etiology and prognosis in post-menopausal women. [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 805.
Abstract 808: Microsatellite instability in metastatic colorectal cancer (mCRC)
Authors: KIM Jee Hung , Kim Chang-gon , Ahn Joong Bae , Jung MinKyu , Beom Seung Hoon , Kim Joo Hoon , Heo Soo Jin , Shin Sang Joon
Published: July 2016

Background: Although early stage colorectal cancer (CRC) with microsatellite instability (MSI) phenotype has a more favorable prognosis, impact on outcome of MSI status in metastatic CRC (mCRC) is rarely known. We aim to explore patterns of metastatic spread and prognosis of CRC with MSI-H (high-level MSI) in comparison to MSI-L (low-level of MSI)/MSS.

Methods: Patients with mCRC who underwent testing for MSI were identified using retrospective cohort of Yonsei Cancer Center. Patients were categorized to MSI status (MSI-H vs. MSS-L/MSS). Patterns of metastatic spread, surgical resection after recurrence and outcome between two groups were compared.

Results: A total of 944 patients were evaluated. Of all, 34 patients (3.6%) had MSI-H tumors. A distinct pattenrs of metastatic spread was observed in MSI-H tumors, namely higher rates of peritoneal metastasis (41% vs 10%, P < 0.001), intraabdominal lymph node metastasis (14.7% vs 4.8%, P = 0.011), and lower rates of lung metastasis (8.8% vs 24.6%, P = 0.034), liver metastasis (20.6% vs 53.6%, P < 0.001). Patients with MSI-H tumors were less likely to receive metastectomy (24.8% vs 55.9%, P < 0.001). Median overall survival was inferior in MSI-H tumor compared with MSI-L/MSS tumor (16.3 months vs 38.1 months, P = 0.005).

Conclusions: Different patterns of metastatic spread and receipt of surgical resection in MSI-H mCRC is demonstrated in this study. In addition, MSI-H phenotype is associated with poorer survival in mCRC on the contrary to early stage disease. Future studies focusing on deeper understanding of tumor biology and therapeutic response in this rare disease entity are warranted.

Citation Format: Jee Hung KIM, Chang-gon Kim, Joong Bae Ahn, MinKyu Jung, Seung Hoon Beom, Joo Hoon Kim, Soo Jin Heo, Sang Joon Shin. Microsatellite instability in metastatic colorectal cancer (mCRC). [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 808.
Abstract 813: The association of androgen metabolizing enzymes and prostate cancer in a multiethnic study
Authors: Grant Delores J. , Howard Lauren , Wiggins Emily , De Hoedt Amanda , Vidal Adriana , Carney Skyla T. , Squires Jill , Magyar Clara E. , Huang Jiaoti , Freedland Stephen J.
Published: July 2016

Introduction: Uridine 5’-diphosphate-glucuronosyltransferase 2B (UGT2B) genes code for enzymes that catalyze the clearance of testosterone, dihydrotestosterone (DHT), and DHT metabolites in the prostate basal and luminal tissue. The expression of the UGT2B15, UGT2B17, and UGT2B28 enzymes has not been evaluated in prostate tissue samples from hormone therapy-naïve patients.

Methods: We determined the expression of the three enzymes in 190 prostate tissue samples from surgical specimens of a multiethnic cohort of patients undergoing radical prostatectomy at the Durham Veterans Affairs Medical Center. The association between each biomarker's percent positive and H-score and the risk of biochemical recurrence (BCR) was tested using separate Cox proportional hazards models. In an exploratory analysis, UGT2B17 total positive and H-score were split at the median and we tested the association between UGT2B17 group and risk of BCR.

Results: The median follow-up was 118 months (IQR: 85-144). We found no association between UGT2B15 or UGT2B28 and risk of BCR. However, there was a trend between UGT2B17 and BCR (HR = 1.01, 95% CI 1.00-1.02, p = 0.11), though not statistically significant. Upon further investigation, we found patients with UGT2B17 total positive above the median had a significant increased risk of BCR on univariable analysis (HR = 1.57, 95% CI 1.02-2.43, p = 0.041) but this association was attenuated in the adjusted model (HR = 1.50, 95% CI 0.94-2.40, p = 0.088).

Conclusions: Our findings suggest that UGT2B17 overexpression was associated with a significant increased risk of BCR. These results are consistent with previous reports that show UGT2B17 significantly expressed in prostate cancer metastases.

Citation Format: Delores J. Grant, Lauren Howard, Emily Wiggins, Amanda De Hoedt, Adriana Vidal, Skyla T. Carney, Jill Squires, Clara E. Magyar, Jiaoti Huang, Stephen J. Freedland. The association of androgen metabolizing enzymes and prostate cancer in a multiethnic study. [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 813.
Abstract 821: Discovery of catechol moiety-containing natural compounds as direct ERK2 inhibitors by in vitro kinase assay and co-crystallography
Authors: Shin Seung Ho , Malakhova Margarita , Kurinov Igor , Lim Do Young , Bode Ann M. , Dong Zigang
Published: July 2016

Catechol (pyrocathechol, 1,2-dihydroxybenzene) is a chemical that is used by industry in the manufacture of certain products. Many natural compounds contain catechol as a functional group found in various fruits and vegetables such as apple, apricot, grape, bananas, soybean, peanut, pear, plum, mango, avocado, and mushroom. Extracellular signal-regulated kinase 2 (ERK2), a protein kinase that belongs to MAPK family, modulates many important functions such as cell growth, apoptosis and transcriptional regulation in cancer. The expression levels of ERK2 mRNA and protein are reportedly very high in many human cancers, including leukemia, colon, breast, lung and skin cancer. Previously, we reported that norathyriol and caffeic acid, which possess the catechol moiety, and catechol itself directly bind and inhibit ERK2 kinase activity. Those findings led us to test catechol moiety-containing natural compounds, luteolin, quercetin, fisetin, 7,3’,4’-trihydroxyisoflavone, and cyanidin, against ERK2 kinase activity. We found that all of these compounds directly bind to ERK2 and inhibit its activity. We further confirmed the results by resolving a co-crystal structure of ERK2 bound with luteolin in the ATP binding site. To apply this finding to cancer, the effect of the compounds was tested on the K562 human myelogenous leukemia cell line where ERK2 is highly expressed and knock-down of the protein reduced anchorage-independent growth. Five newly-found ERK2 inhibitors (luteolin, quercetin, fisetin, 7,3’,4’-trihydroxyisoflavone and cyanidin) and three known ERK2 inhibitors (norathyriol, caffeic acid and catechol) decreased cell viability of K562 at 40 ìM. When the eight compounds were used at the same dose in combination (5 ìM each and 40 ìM total), the mixture inhibited ERK2 and reduced cell viability of K562 cells. In summary, using in vitro kinase assays and co-crystallography, we identified new ERK2 inhibitors that contain the catechol functional group, and showed anti-cancer effects of the compounds by cell viability assay and soft agar assay. This work impacts the cancer prevention community by showing that many natural compounds can work together targeting ERK2, an important target in cancer.

Citation Format: Seung Ho Shin, Margarita Malakhova, Igor Kurinov, Do Young Lim, Ann M. Bode, Zigang Dong. Discovery of catechol moiety-containing natural compounds as direct ERK2 inhibitors by in vitro kinase assay and co-crystallography. [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 821.
Abstract 826: Benzyl isothiocyanate inhibits breast cancer-induced osteoclastogenesis
Authors: Pore Subrata K. , Sehrawat Anuradha , Singh Shivendra V.
Published: July 2016

Breast cancer is a major public health issue for women worldwide and 70% of the disease preferentially metastasizes to bone resulting in its destruction. Advanced breast cancer releases osteoclastogenic factors to promote bone resorption. As bone metastasis with breast cancer is associated with high mortality, osteoclastogenesis is a potential therapeutic target. We have shown previously that benzyl isothiocyanate (BITC), which is present in edible cruciferous vegetables, inhibits breast cancer development in a transgenic mouse model. The present study was designed to determine effect of BITC on breast cancer-induced osteoclastogenesis. We employed well-established in vitro osteoclast co-culture system of Raw 264.7 murine macrophage cells with human breast cancer cells for this assessment. BITC treatment significantly inhibited the number as well as size of osteoclasts in a dose dependent manner in every co-culture experiment. In addition, BITC downregulated both mRNA and protein levels of macrophage colony stimulating factor (MCSF) and receptor activator of NF-êB ligand (RANKL) which are key factors for formation of osteoclasts. Moreover, receptor activator of NF-êB ligand (RANKL)-induced osteoclast differentiation as well as bone resorption was suppressed in the presence of BITC. On the other hand, BITC induced osteoprotegerin (OPG) acting as a decoy receptor of RANKL and negatively regulated osteoclast formation. Quantitative PCR analysis revealed that mRNA levels of osteoclastogenesis-related cytokines and proteins (IL-1â, IL-6, TNF-á, GM-CSF, OPG, RUNX2) were repressed in BITC-treated breast cancer cells. Taken together, BITC showed inhibitory activity on breast cancer-mediated osteoclastogenesis. This study was supported by the grant CA129347 awarded by the National Cancer Institute.

Citation Format: Subrata K. Pore, Anuradha Sehrawat, Shivendra V. Singh. Benzyl isothiocyanate inhibits breast cancer-induced osteoclastogenesis. [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 826.
Abstract 851: Analysis of cancer-initiating mosaic mutation in germline samples of pediatric cancer patients by next generation sequencing
Authors: Ma Xiaotu , Yergeau Donald , Vadodaria Bhavin , Nakitandwe Joy , Easton John , Shurtleff Sheila , Zhang Jinghui
Published: July 2016

A cancer predisposition mutation can occur during post-zygotic stages resulting in a mosaic mutation pattern across tissues. Mosaic germline mutations such as those affecting RB1, TP53 and AKT1 are important for clinical testing. However, it is challenging to detect mosaic mutation by genome-wide sequencing approaches as a mutant allele present at a low fraction in a germline sample can be caused by either tumor-in-normal contamination or mosaicism. Here we propose a two-step analytical approach to distinguish between these two possibilities. The first involves assessing the level of tumor-in-normal contamination using all clonal somatic mutations identified through the analysis of tumor purity and chromosome ploidy. The second step determines the probability of mosaicism for a candidate variant by calculating the binomial probability of its mutant allele fraction (MAF) under the contamination hypothesis. We applied this process to 1,172 paired tumor-normal pediatric cancer samples analyzed by whole-genome or whole-exome sequencing. Six mosaic variants were identified, including three pathogenic TP53 mutations with a mutant allele fractions (MAF) ranging from 0.2 to 0.3, one RB1 mutation in a retinoblastoma case with a MAF of 0.08, one IDH1 p.R132H mutation in an acute myeloid leukemiaý case with a MAF of 0.21, and one mosaic RYR2 p.E1690D mutation in an osteosarcoma case with a MAF of 0.11. At the same time, we were able to reject three candidate TP53 variants caused by contamination with MAFs ranging from 0.06 to 0.33. All predicted mosaic mutations were verified by ultra deep sequencing at 10,000X coverage. This experience demonstrates the feasibility of detecting germline mosaicism from whole-genome or whole-exome sequencing of paired tumor-normal samples and would caution accurate analysis of tumor-in-normal contamination when evaluating candidate germline mosaicism.

Citation Format: Xiaotu Ma, Donald Yergeau, Bhavin Vadodaria, Joy Nakitandwe, John Easton, Sheila Shurtleff, Jinghui Zhang. Analysis of cancer-initiating mosaic mutation in germline samples of pediatric cancer patients by next generation sequencing. [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 851.
Abstract 882: TMPRSS2-ERG drives global mistargeting of mammalian SWI/SNF (BAF) complexes in prostate cancer
Authors: Sandoval Gabriel J. , Pulice John L. , Takeda David Y. , Schenone Monica A. , Pop Marius , Boulay Gaylor , Rivera Miguel N. , Ronco Lucienne , Hahn William C. , Kadoch Cigall
Published: July 2016

Prostate cancer remains one of the leading causes of cancer-related death in men. Chromosomal rearrangements resulting in the fusion of the androgen regulated gene TMPRSS2 and the ETS-family transcription factor ERG occur in over 50% of all prostate cancer cases. Recent studies enabled by genome-wide methodologies have implicated altered epigenomic landscapes and changes in DNA accessibility as major contributors to ERG-driven oncogenesis, however the precise mechanism underlying the ERG transcriptional signature has to date remained unclear. Here we performed the first endogenous purification and SILAC-mass spectrometric analysis of ERG in TMPRSS2-ERG prostate cancer cells. Remarkably, we demonstrate that ERG directly interacts with the mammalian SWI/SNF (BAF) ATP-dependent chromatin remodeling complex, which was recently shown to be mutated in >20% of human malignancies. ERG co-localizes with BAF complexes genome-wide, resulting in specific ERG-dependent BAF complex targeting to sites enriched in known ERG, FOXA1, and HOXB13 motifs; additionally, loss of ERG in TMPRSS2-ERG driven cell lines results in dramatic retargeting of BAF complexes away from ERG-dependent sites, to sites enriched in known AR and CTCF motifs. Importantly, ERG-driven BAF complex retargeting contributes to activation of TMPRSS2-ERG prostate cancer gene expression signatures. We map the ERG-BAF interaction to a specific region within the ERG amino acid sequence and find that this region is required to bind BAF complexes. These studies reveal a novel, unexpected mechanism of action of ERG-driven oncogenesis and offers new strategies for therapeutic intervention.

Citation Format: Gabriel J. Sandoval, John L. Pulice, David Y. Takeda, Monica A. Schenone, Marius Pop, Gaylor Boulay, Miguel N. Rivera, Lucienne Ronco, William C. Hahn, Cigall Kadoch. TMPRSS2-ERG drives global mistargeting of mammalian SWI/SNF (BAF) complexes in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 882.
Abstract 89: Whole-exome sequencing and immune profiling of early-stage lung adenocarcinoma
Authors: Kadara Humam , Choi Murim , Zhang Jiexin , Parra Cuentas Edwin , Rodriguez Canales Jaime , Gaffney Stephen , Zhao Zi-Ming , Behrens Carmen , Fujimoto Junya , Chow Chi-Wan , Kalhor Neda , Moran Cesar , Rimm David , Swisher Stephen G. , Gibbons Don L. , Heymach John V. , Kaftan Edward , Townsend Jeffrey , Lynch Thomas J. , Schlessinger Joseph , Lee J. Jack , Lifton Richard , Wistuba Ignacio I. , Herbst Roy S.
Published: July 2016

PURPOSE: Lung adenocarcinomas (LUADs) lead to the preponderance of deaths attributable to lung cancer. We performed whole-exome sequencing (WES), comprehensive immune profiling and clinicopathological analysis of LUADs to better understand the molecular pathogenesis of this disease and identify clinically relevant molecular markers.

METHODS: We performed WES of 108 paired surgically resected stage I-III LUADs and normal lung tissues using the Illumina HiSeq 2000 platform. Additionally, ten immune related markers (PD-L1, PD-1, CD3, CD4, CD8, CD45ro, CD57, CD68, FOXP3 and Granzyme B) were profiled by imaging-based immunohistochemistry in a subset of LUADs (n = 92). Associations among mutations, immune markers and clinicopathological variables were analyzed using ANOVA and Fishers Exact tests. Cox proportional hazards regression models were employed for multivariate analysis of clinical outcome.

RESULTS: LUADs in this cohort exhibited an average of 243 coding mutations per tumor. We identified 28 genes with significant enrichment for mutation. SETD2-mutant LUADS exhibited relatively poor recurrence-free survival (RFS) and mutations in STK11 and ATM were associated with poor RFS in KRAS-mutant tumors. EGFR, KEAP1 and PIK3CA mutations were predictive of poor response to adjuvant therapy. Immune marker analysis demonstrated that PD-L1 expression was increased in smoker compared to non-smoker LUADs and, along with other immune markers, was positively correlated with somatic mutation burden. Moreover, immune marker levels including PD-L1 were elevated in TP53-mutant LUADs. In contrast, STK11 and U2AF1 mutant tumors exhibited a suppressed immune response and LUADs with PIK3CA mutations exhibited markedly decreased tumoral PD-L1 expression.

CONCLUSION: Our study highlights mutations that may impact clinical outcome and personalized strategies for immune-based therapy of early-stage LUAD patients.

Citation Format: Humam Kadara, Murim Choi, Jiexin Zhang, Edwin Parra Cuentas, Jaime Rodriguez Canales, Stephen Gaffney, Zi-Ming Zhao, Carmen Behrens, Junya Fujimoto, Chi-Wan Chow, Neda Kalhor, Cesar Moran, David Rimm, Stephen G. Swisher, Don L. Gibbons, John V. Heymach, Edward Kaftan, Jeffrey Townsend, Thomas J. Lynch, Joseph Schlessinger, J. Jack Lee, Richard Lifton, Ignacio I. Wistuba, Roy S. Herbst. Whole-exome sequencing and immune profiling of early-stage lung adenocarcinoma. [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 89.
Abstract 914: Sox10 regulates stem- and mesenchymal-like features in mammary cells
Authors: Dravis Christopher , Wahl Geoffrey M.
Published: July 2016

To discover mechanisms that mediate the initiation and progression of aggressive and stem-like breast cancers, we characterized signaling networks that are present in the mammary stem cells responsible for fetal and adult mammary development. These analyses identified a signaling axis between FGF signaling and the transcription factor, Sox10. Here we report that Sox10 is specifically expressed in mammary cells that exhibit the highest levels of stem/progenitor activity. This includes fetal and adult mammary cells in vivo and mammary organoids in vitro. Sox10 is functionally relevant, as its deletion reduces stem/progenitor competence, while its overexpression increases stem/progenitor activity. Intriguingly, we also discover that Sox10 overexpression elicits epithelial-to-mesenchymal transition (EMT) in mammary organoids in an FGF signaling-dependent manner. Further, modulation of Sox10 levels can induce sequential EMT, migratory, and clonogenic behaviors that strikingly resemble proposed mechanisms of metastasis. Consistent with these findings, we report that Sox10 is preferentially expressed in the most stem- and EMT-like triple negative breast cancer subtypes, which suggest that Sox10 may reprise these same functions during tumorigenesis. Indeed, we find that in an autochthonous mouse model of basal-like breast cancer, Sox10 is expressed at high levels and that these Sox10+ tumor cells exhibit characteristics that are similar to mammary cells in which Sox10 is ectopically expressed. Collectively, these results demonstrate a signaling mechanism through which stem and mesenchymal-like states are acquired in mammary cells, and indicate possible therapeutic targets to counter these functions in breast cancers for which targeted therapies are currently unavailable.

Citation Format: Christopher Dravis, Geoffrey M. Wahl. Sox10 regulates stem- and mesenchymal-like features in mammary cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 914.
Abstract 924: Upregulation of the MET transcript is consistently associated with invasion and tumor budding in colorectal cancer
Authors: Bradley Conor , Dunne Philip , McQuaid Stephen , Bingham Victoria , Lawler Mark , Salto-Tellez Manuel , Johnston Patrick , Van Schaeybroeck Sandra
Published: July 2016

Background:

The c-MET proto-oncogene is frequently overexpressed (50-60%), amplified (1-3%), and mutated (1-3%) in colorectal cancer (CRC). Hepatocyte growth factor (HGF)-dependent and independent activation of c-MET has been associated with increased survival and resistance to targeted therapies. This study aimed to investigate the role of the HGF/c-MET axis in regulating migration/invasion in CRC, using pre-clinical models and clinical samples.

Methods:

In order to model CRC tumour cell invasion, we have generated invasive CRC subpopulations using Boyden Invasion chambers. To model the CRC microenvironment, we have used a range of co-culture techniques with CRC cells and colon fibroblasts. Migration/invasion was determined using xCELLigence System (Roche). c-MET expression in parental and invasive cell lines was measured using Western blotting and qRT-PCR. c-MET expression in CRC FFPE tissues was measured using IHC and RNAScope®.

Results:

We identified marked upregulated expression of c-MET at both the protein and transcript levels in our invasive CRC cell line models. Importantly, both parental and invasive subpopulations were found to be inherently dependent on c-MET for migration, as RNAi against c-MET abrogated migration/invasion in both parental and invasive models. We also demonstrated that stimulation of CRC cells with rh-HGF resulted in increased CRC cell migration/invasion. In addition, co-culture of CRC cells with colonic myofibroblasts, resulted in marked increases in migratory and invasive capacity, and this was dependent on HGF/c-MET signaling. Interestingly, stimulation with myofibroblast conditioned medium or HGF promotes rapid degradation of c-MET at the protein level, followed by recycling, while MET transcript remains unaltered, illustrating a dynamic expression of c-MET protein in response to activation. We further showed that MET is transcriptionally upregulated in tumour budding foci at the invasive front of a cohort of stage III CRC tumors. Intriguingly, c-MET protein levels do not correlate with the transcript, most likely due to a similar protein degradation process observed in our aforementioned in vitro models.

Conclusions:

We show for the first time a key role for transcriptional upregulation of MET as a molecular driver of tumour invasion, both in vitro and in stage III CRC tumours.

Citation Format: Conor Bradley, Philip Dunne, Stephen McQuaid, Victoria Bingham, Mark Lawler, Manuel Salto-Tellez, Patrick Johnston, Sandra Van Schaeybroeck. Upregulation of the MET transcript is consistently associated with invasion and tumor budding in colorectal 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 924.
Abstract 932: AR signaling differentially regulates the growth of prostate epithelial cells
Authors: Yu Xiuping , Yang Shu
Published: July 2016

The prostate is an androgen-dependent organ. Androgen stimulates the growth of normal prostatic cells, as well as prostate cancer (PCa). However, AR signaling does not always promote cell proliferation, in some cases, it may even inhibit the growth of prostatic cells. In this study, we used NHPrE, a cell line derived from normal human prostate epithelia, to study how AR signaling modulates the growth of prostatic cells and to explore the mechanisms involved in AR's differential function in vitro and in vivo. Our results showed that inducing AR signaling in NHPrE cells suppressed their growth in vitro, concomitant with a reduction of Myc. In contrast, ectopic expression of AR in NHPrE cells induced the expression of Myc and pSTAT3 and drove the development of invasive PCa in vivo, suggesting a distinct in vivo function of AR signaling in prostatic cells. Furthermore, our data showed that the presence of prostate stromal cells over-rode androgen-mediated growth inhibition on NHPrE/AR cells in vitro; and IL-6/pSTAT3 was implicated in this stromal/epithelial communication. In conclusion, our study showed that AR signaling differentially regulated the growth of prostatic cells in vitro and in vivo through a mechanism that involved stromal/epithelial interactions.

Citation Format: Xiuping Yu, Shu Yang. AR signaling differentially regulates the growth of prostate epithelial cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 932.
Abstract 958: MiR-196b is an epigenetically silenced tumor suppressor for lung cancer
Authors: Tellez Carmen S. , Juri Daniel E. , Do Kieu , Picchi Maria A. , Wang Teresa , Liu Gang , Spira Avrum , Belinsky Steven A.
Published: July 2016

Silencing of microRNAs by promoter hypermethylation could play a significant role in lung cancer initiation and progression. A pre-malignancy model using carcinogen transformed human bronchial epithelial cells (HBECs) treated with the demethylating agent 5-aza-2’deoxycytidine followed by next-generation sequencing identified miR-196b and miR-34c-5p as being epigenetically regulated. Bisulfite sequencing confirmed silencing via dense promoter hypermethylation that was commonly replicated in malignant cell lines and primary tumors. Functional studies of miR-196b revealed that it was epigenetically silenced by chromatin modification and DNA methylation during transformation of HBECs and re-expression modulated genes involved in cell cycle regulation. Stable re-expression led to a cell cycle arrest mediated in part through transcriptional regulation of the FOS oncogene that was replicated in vivo by a profound reduction in growth of tumor xenografts. Luciferase assays demonstrated that forced expression of miR-196b inhibits FOS promoter and AP-1 reporter activity. A case-control study showed that methylation of miR-196b in sputum was strongly associated with lung cancer (OR = 4.7, p<0.001). These studies identify miR-196 as a tumor suppressor whose silencing early in lung carcinogenesis may provide a selective growth advantage to pre-malignant cells. Targeted delivery of this microRNA could benefit prevention and therapy for the management of lung cancer.

Citation Format: Carmen S. Tellez, Daniel E. Juri, Kieu Do, Maria A. Picchi, Teresa Wang, Gang Liu, Avrum Spira, Steven A. Belinsky. MiR-196b is an epigenetically silenced tumor suppressor for lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 958.
Abstract 963: Epigenetic silencing of miR-199a-3p is associated with increased cell survival, enhanced cell motility, and sensitivity to tivantinib through regulation of c-Met expression in hepatocellular carcinoma
Authors: Yasui Kohichiroh , Tomie Akira , Iwai Naoto , Kitaichi Tomoko , Dohi Osamu , Gen Yasuyuki , Itoh Yoshito
Published: July 2016

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and can act as tumor suppressors or oncogenes. To identify miRNA involved in the development of HCC, we performed a genome-wide miRNA gene expression analysis using the human miRNA microarray (Agilent) in paired tumor and non-tumor tissues from patients with primary HCC. The array-based miRNA expression profiles were validated by quantitative PCR. We also screened for genes with promoter DNA hypermethylation using a genome-wide DNA methylation microarray analysis (the Illumina HumanMethylation27 BeadChip) in primary HCCs. We found that miR-214-3p, miR-199a-5p, and miR-199a-3p were significantly down-regulated by aberrant promoter hypermethylation in HCC tumors. The c-Met gene was identified as the direct target of miR-199a-3p. Our study showed that reduced expression of miR-199a-3p was associated with increased cell survival through the c-Met/Akt pathway and enhanced cell motility through phosphorylation of FAK. The transfection of miR-199a-3p mimic reduced the expression of c-Met and enhanced the sensitivity to the c-Met inhibitor tivantinib in HCC cells. In conclusion, our results suggest that epigenetic silencing of tumor suppressor miR-199a-3p may be involved in hepatocarcinogenesis through regulation of c-Met expressionand and be associated with the sensitivity to tivantinib.

Citation Format: Kohichiroh Yasui, Akira Tomie, Naoto Iwai, Tomoko Kitaichi, Osamu Dohi, Yasuyuki Gen, Yoshito Itoh. Epigenetic silencing of miR-199a-3p is associated with increased cell survival, enhanced cell motility, and sensitivity to tivantinib through regulation of c-Met expression in hepatocellular carcinoma. [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 963.