bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4399: Hypoxia as a driver of myeloid-derived suppressor cell recruitment in hepatocellular carcinoma via CCL26/CX3CR1
Authors: Chiu David Kung-Chun , Xu Iris Ming-Jing , Lai Robin Kit-Ho , Tse Aki Pui-Wah , Wei Larry Lai , Koh Hui-Yu , Lo Regina Cheuk-Lam , Wong Chun-Ming , Ng Irene Oi-Lin , Wong. Carmen Chak-Lui
Published: July 2016

Background and Objective: Myeloid-derived suppressor cells (MDSCs) accumulate in tumors and highly pro-tumorigenic. These MDSCs exhibit inhibitory functions against effector T cells and natural killer cells in tumor sites, as well as secrete pro-angiogenic factors or differentiate to endothelial cells to promote angiogenesis and metastasis. While it is appreciated that depletion of MDSCs could bring tumoricidal effects, there are significant gaps in knowledge about the underlying mechanisms responsible for MDSC recruitment to tumor sites. Hypoxia, O2 deprivation, is an important factor in the tumor microenvironment of HCC that modifies the stromal components. Using hepatocellular carcinoma (HCC) as a model, we investigated whether hypoxia is a driver of MDSC recruitment in HCC.

Experimental Procedures: Gene profiling of HCC cells exposed to hypoxia and normoxia were analyzed by transcriptome sequencing to identify potential hypoxia-induced chemokines for MDSC recruitment. MDSCs were isolated from HCC-bearing mice by magnetic bead sorting for different functional assays. Boyden chambers were used to evaluate the invasive ability of MDSCs. Flow cytometry was used to detect the frequencies of tumor-associated MDSCs in orthotopic HCC mouse models.

Results: We observed that MDSCs preferentially infiltrated into hypoxic regions in human HCC tissues and hypoxia-induced MDSC infiltration was dependent on hypoxia-inducible factors (HIFs). HIFs activated the transcription of chemokine (C-C motif) ligand 26 (CCL26) in HCC cells to recruit chemokine (C-X3-C motif) receptor 1 (CX3CR1)-expressing MDSCs to the primary tumor. Knockdown of CCL26 in HCC cells profoundly reduces MDSC recruitment, angiogenesis, and tumor growth. Therapeutically, blockade of CCL26 production in HCC cells by HIF inhibitor, digoxin, or blockade of CX3CR1 in MDSCs by CX3CR1 neutralizing antibody could substantially suppress MDSC recruitment and tumor growth.

Conclusion: This study unprecedentedly reveals a novel molecular mechanism by which HCC cells direct MDSC homing to primary tumor and suggests that targeting MDSC recruitment represents an attractive therapeutic approach against HCC.

Citation Format: David Kung-Chun Chiu, Iris Ming-Jing Xu, Robin Kit-Ho Lai, Aki Pui-Wah Tse, Larry Lai Wei, Hui-Yu Koh, Regina Cheuk-Lam Lo, Chun-Ming Wong, Irene Oi-Lin Ng, Carmen Chak-Lui Wong. Hypoxia as a driver of myeloid-derived suppressor cell recruitment in hepatocellular carcinoma via CCL26/CX3CR1. [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 4399.
Abstract 4401: Disclosing the Hippo signaling networks in castration-resistant prostate cancer
Authors: Ready Damien , Yildiz Yuksel , Funari Vincent , Bozdag Serdar , Cinar Bekir
Published: July 2016

Prostate cancer often evolves as a metastatic castration-resistant disease, which is lethal. Despite recent advances, the molecular mechanisms contributing to this lethal disease are largely unknown. Our initial studies have indicated that the loss-of-function of the STK4-encoded MST1 protein kinase, a key component of the Hippo tumor suppressor pathway, may play a prominent role in the etiology of prostate cancer progression. Here, we establish cytoplasmic-, membrane- or nuclear-localized MST1 cell models to elucidate a possible mode of actions by which MST1 control the disease progression. We show that membrane or nuclear MST1 confers superior growth inhibitory effects on castration-resistant prostate cancer cells ex vivo and in vivo in comparison with cytoplasmic MST1, though they express the similar levels of MST1 protein. Consistently, a number of differentially expressed genes (2-fold with FDR ≤ 0.05) regulated by membrane or nuclear MST1 protein are much greater than cytoplasmic MST1, as assessed by RNA-sequencing and bioinformatics analysis. Functional annotation clustering reveals that genes affected by cytoplasmic MST1 protein are mostly associated with cell-cell interactions and membrane trafficking. However, expression of genes altered by membrane or nuclear MST1 involves much broader cellular biology that includes cell signaling, steroid biogenesis, lipid and cholesterol metabolisms, and developmental processes. In addition, Ingenuity Pathway analysis indicates that several genes effected by ectopic MST1 protein the defined cell location interact with androgen receptor pathway signaling, a critical regulator of prostate cancer pathogenesis. The results of this study suggest that the Hippo pathway signaling restricts prostate cancer progression by modulating the array of signaling networks, indicating important therapeutic implications for human disease.

Citation Format: Damien Ready, Yuksel Yildiz, Vincent Funari, Serdar Bozdag, Bekir Cinar. Disclosing the Hippo signaling networks in castration-resistant 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 4401.
Abstract 4402: Glycosyltransferase ST6Gal-I enables cancer cells to survive upon growth factor deprivation
Authors: Britain Colleen , Bellis Susan
Published: July 2016

Tumor growth often outpaces the tumor's ability to form new vascularization leading to a tumor microenvironment containing areas with limited access to serum-derived growth factors (GFs). How tumor cells are able to proliferate and survive in the absence of GFs is not completely understood. Recent work from our group has uncovered a novel mechanism by which ST6Gal-I, a glycosyltransferase upregulated in many cancers, enables cells to evade GF deprivation-mediated apoptosis. ST6Gal-I is a trans-Golgi sialyltransferase that catalyzes the addition of a bulky, negatively charged sialic acid in an α2-6 manner to the termini of N-linked glycans on glycoproteins bound for the plasma membrane. This sialic acid addition can modulate protein conformation and function, similar to phosphorylation. Prior studies from our group show that ST6Gal-I activity confers a cancer stem cell phenotype as well as enhances chemoresistance, leading us to hypothesize that ST6Gal-I promotes tumor cell survival, invasion and metastasis. To better understand the role ST6Gal-I plays in tumor cell survival upon GF deprivation, we monitored the response of ovarian and pancreatic cancer cells with either ST6Gal-I overexpression or knockdown to serum starvation. Upon serum starvation, cells overexpressing ST6Gal-I preferentially survived and continued to proliferate whereas cells with ST6Gal-I knockdown underwent cell death more readily, as evidenced by live-dead cell staining and cell proliferation assays. Furthermore, in the absence of serum, ST6Gal-I activity promotes the activation and/or expression of multiple pro-survival proteins, such as Akt, p70-S6K, NFκB, and cIAP2, as well as cell cycle regulators including CyclinD2. Interestingly, upon long-term GF deprivation, immunoblotting analysis reveals a selective enrichment for ST6Gal-I in the remaining live cells providing evidence that cells with high ST6Gal-I expression selectively survive GF deficiency. Collectively these data suggest that upregulation of ST6Gal-I in tumor cells may promote cell survival in areas of the tumor microenvironment that have decreased access to a blood supply.

Citation Format: Colleen Britain, Susan Bellis. Glycosyltransferase ST6Gal-I enables cancer cells to survive upon growth factor deprivation. [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 4402.
Abstract 4404: NFAT-5/STAT-3 interaction mediates synergism of high salt with IL-17 towards induction of VEGF-A expression in breast cancer cells
Authors: Amara Suneetha , Alotaibi Dalal , Tiriveedhi Venkataswarup
Published: July 2016

Chronic inflammation has been considered an important player in cancer proliferation and progression. High salt (sodium chloride) has been considered a potent inducer of chronic inflammation. In this study, we investigated the synergistic role of high salt with IL-17 towards induction of inflammatory and angiogenic stress factor VEGF. Stimulation of MCF-7 breast cancer cells with high salt (0.2 M NaCl) and sub-minimal IL-17 (1 ng/mL) enhanced the expression of VEGF-A (2.9 and 2.6 fold, respectively p<0.05) compared to untreated cells. Further, co-treatment with both high salt and sub-minimal IL-17 demonstrated a 5.9 fold increase in VEGF-A expression (p<0.01), thus suggesting a synergistic role of these both factors. VEGF-A promoter analysis and specific siRNA knock-down of transcription factors revealed that high salt induces VEGF-A through NFAT5, while IL-17 induced VEGF-A via STAT-3 signaling mechanisms. Treatment of the supernatant from activated MCF-7 cells on normalized human aortic endothelial cells induced enhanced cell migration and expression of migration specific factors VCAM, β1-integrin and CD31. Based on these data, we conclude that high salt synergizes with pro-inflammatory IL-17 to potentially induce cancer progression and metastasis through VEGF-A expression. Therefore, low salt diet, anti-NFAT5 and anti-STAT3 therapies might provide novel venues for enhanced efficiency of current cancer therapy.

Citation Format: Suneetha Amara, Dalal Alotaibi, Venkataswarup Tiriveedhi. NFAT-5/STAT-3 interaction mediates synergism of high salt with IL-17 towards induction of VEGF-A expression in breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4404.
Abstract 4413: Thrombospondin-1 is a master regulator of glioblastoma vascularization and infiltration
Authors: Daubon Thomas , Leon Celine , Clarke Kim , Poulet Mathilde , Miletic Hrvoje , Falciani Francesco , Bjerkvig Rolf , Bikfalvi Andreas
Published: July 2016

Glioblastoma are heterogeneous tumours composed of different subpopulations of cells with different behavioral characteristics. Areas of highly angiogenic cells co-exist with populations of non-angiogenic but highly invasive and infiltrative cells. This heterogeneity is a key clinical challenge in glioma treatment.

We used a large scale RNA sequencing approach to investigate the molecular components which differentiate infiltrative from angiogenic cells, in a Patient Derived Xenograft (PDX) mouse model. Our bioinformatic analysis revealed TGFβ1 to be a master regulator of tumor development, and Thrombospondin-1 (Tsp1) to be up regulated in infiltrative areas as compared to angiogenic area. Thrombospondins are known as anti-angiogenic factors, however the full roles of these multi domain proteins in tumor development remain to be elucidated.

We found Tsp1 expression to be upregulated in grade IV-GBM and in silico in the GBM mesenchymal subclass. It is expressed not only in tumor cells, but also in tumor blood vessel endothelial cells (ECs). TGFβ1 transcriptionally regulated Tsp1 via Smad1 and Smad3. However, contrary to previous results, we found that Tsp1 was not involved in TGFβ1-activation in tumor cells. We showed that Tsp1 regulates cell migration and invasion both in vitro and in vivo. Inhibition of Tsp1 expression in vivo correlated with increased tumor vascularization in both the chick CAM assay and the PDX mouse model.

Anti-angiogenic treatments in the PDX mouse model leads to increased tumor hypoxia and invasive tumor cell behavior, as described in our previous work. In this context, both TGFβ1 and Tsp1 were upregulated in tumor cells. Downregulation of tumor-derived Tsp1 by shRNA in the presence of anti-angiogenic therapy led to reduced tumor growth and invasion in vivo. Finally, peptide-mediated inhibition of Tsp1 activity demonstrated that Tsp1/CD47 interaction is involved in the invasive capacity of GBM cells.

Taken together, our data suggest that Tsp1 inhibition may be a promising therapeutic approach to limit tumor infiltration induced by treatment with anti-angiogenic agents.

Citation Format: Thomas Daubon, Celine Leon, Kim Clarke, Mathilde Poulet, Hrvoje Miletic, Francesco Falciani, Rolf Bjerkvig, Andreas Bikfalvi. Thrombospondin-1 is a master regulator of glioblastoma vascularization and infiltration. [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 4413.
Abstract 4416: Combination of HSP90 and proteasome inhibitor is effective in pancreatic cancer
Authors: Nagaraju Ganji Purnachandra , Rajitha Balney , Benton Leah , El-Rayes Bassel F.
Published: July 2016

Background: Heat shock protein (HSP90) and proteasome play an important role in cellular protein trafficking and degradation in pancreatic cancer (PC). Given the overlap in the mechanism of action, we investigated the effects of the combination of pharmacological inhibitors of HSP90 (Ganetespib) and proteasome (Carfilzomib) on PC cells in vitro and in vivo.

Methods: The combined effects of ganetespib and carfilzomib were evaluated in MIA PaCa-2 and HPAC cell lines using a cell proliferation

assay. Effects on the expression of survival (PI3K/AKT, and ERK), cell cycle (Cdc-2 and cyclin B1), angiogenesis (HIF-1α and VEGF) and autophagy (LC3 A/B, and Atg7) pathways were examined by Western blot. Cell cycle analysis was performed by FACS assay. HPAC cell lines were tested for efficacy to ganetespib, carfilzomib, alone and in combination, using an in vivo tumor xenograft model.

Results: The combination of ganetespib and carfilzomib significantly decreased cell proliferation, induced G2-M cell cycle arrest and induced autophagy in both MIA PaCa-2 and HPAC cell lines. Ganetespib and carfilzomib also decreased the activation of ERK, PI3K/AKT, and autophagy signaling molecules (LC3 A/B, and Atg7). In animal models, ganetespib potentiated the effects of carfilzomib, as measured by tumor

volume. Western blot analysis from tumors removed from animals confirmed the effects of ganetespib and carfilzomib on survival, cell cycle, autophagy and angiogenesis.

Conclusion: These observations provide preclinical proof-of-principle that combinatorial targeting of HSP90 and proteasome is a rational approach for development in PC clinical trials.

Citation Format: Ganji Purnachandra Nagaraju, Balney Rajitha, Leah Benton, Bassel F. El-Rayes. Combination of HSP90 and proteasome inhibitor is effective in pancreatic 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 4416.
Abstract 4419: Exploration of alternative pathways mediated by IFNL4 and related to cell proliferation and death in a hepatoma cell line
Authors: Wang Fang , Onabajo Olusegun O. , Rao Nina , Prokunina-Olsson Ludmila
Published: July 2016

The ability to generate interferon lambda 4 (IFNL4), the most recently discovered member of the type-III interferon family, has been associated with impaired clearance of hepatitis C virus (HCV). IFNL4 signals through an IFNL receptor complex that serves all type-III interferons and consists of receptors IFNLR1 and IL10R2. Signaling through IFNL receptor complex leads to activation of the JAK-STAT pathway and transcriptional activation of interferon-stimulated genes (ISG). Intracellular expression of IFNL4 also causes reduced proliferation and increased cell death in human hepatic cells but it remains unclear if this is a result of JAK-STAT signaling through IFNL receptor complex or through some other alternative mechanisms. To address this question, we used CRISPR-CAS9 system to knock-out IFNLR1 in a hepatoma HepG2 cells. We designed and tested six guide RNAs (gRNAs) for IFNLR1 exons. CAS9 protein and gRNAs that most efficiently induced mutations in IFNLR1 were introduced into HepG2 cells to generate stable cell lines. No obvious defects were observed in IFNLR1-KO-HepG2 cells, indicating that loss of IFNLR1 had no deleterious effects on cell viability. We selected three stable clones which had sequencing-confirmed inactivating IFNLR1 mutations and Western blot-confirmed lack of IFNLR1 protein expression. Compared to the parental HepG2 cells the IFNLR1-KO-HepG2 cells were deficient in the ability to induce the luciferase-tagged interferon-stimulated response element (ISRE-Luc) reporter after treatment with recombinant IFNL4 and IFNL3 proteins, which signal through the same receptor complex. At the same time, response to IFNa treatment was not affected because IFNa signals through its own receptor complex. These results indicate that, as other type-III interferons, IFNL4 induces ISRE signaling through IFNL receptor complex. INFLR1-KO-HepG2 cells provide a highly efficient model for studies of factors involved in signaling of type-III interferons, including IFNL4. Next, we will investigate alternative pathways involved in anti-proliferative and cell death-related phenotypes induced by IFNL4 in HepG2 cell. We will perform RNA-seq on parental and IFNLR1-KO-HepG2 cells treated with or without IFNL4, IFNL3 and IFNa. These results will contribute to a better understanding of IFNL4 function, which can be relevant for viral infections and cancer.

Citation Format: Fang Wang, Olusegun O. Onabajo, Nina Rao, Ludmila Prokunina-Olsson. Exploration of alternative pathways mediated by IFNL4 and related to cell proliferation and death in a hepatoma cell line. [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 4419.
Abstract 4424: Acetylation of Fascin on the lysine 471 residue produces a change in growth and mobility phenotype of esophageal cancer cells by inducing F-actin rearrangement
Authors: Zeng Fa-Min , Wang Xiao-Ning , Zhang Ying-Li , Liao Lian-Di , Li En-Min , Xu Li-Yan
Published: July 2016

Elevated expressions of fascin mRNA and protein in cancer cells have been correlated with aggressive clinical course, poor prognosis and shorter survival. Fascin is an evolutionarily conserved actin-binding protein that plays a key role in forming filopodia. It is reported that the lysine 471 residue (K471) of fascin can be acetylated and also involves in its actin binding, but the roles of K471 acetylation in filopodia formation are still uncertain. In this paper, we produced point mutants mimicking the active deacetylated (Lys471Arg, K471R) or inactive acetylated (Lys471Gln, K471Q) states of fascin. The actin-binding assay showed that the fascin K471Q mutation decreased the actin binding activity of fascin, compared with wild-type fascin (WT) and K471R mutant fascin in the esophageal cancer cells. Moreover, live imaging revealed that K471Q mutant fascin reduced the number and length of filopodia, whereas the K471R mutant increased filopodia frequency. The fascin K471Q mutation changed the straight filopodia became forniciform filopodia. The protrusion presented abnormal morphology when cells spread. Further functional experiments showed that the K471Q mutant fascin rendered the tumor cell migration, invasion and tumorigenesis decrease significantly, whereas K471R mutant fascin like WT fascin still maintained a strong ability. Taken together, we propose that the lysine K471 residue may be a key specific actin crosslinking site and also involved in the tumor biological function of fascin.

Citation Format: Fa-Min Zeng, Xiao-Ning Wang, Ying-Li Zhang, Lian-Di Liao, En-Min Li, Li-Yan Xu. Acetylation of Fascin on the lysine 471 residue produces a change in growth and mobility phenotype of esophageal cancer cells by inducing F-actin rearrangement. [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 4424.
Abstract 443: Prognostic significance of CXCL12 protein levels in high-grade serous ovarian carcinoma
Authors: Buisseret Laurence , Delisle Vincent , Communal Laudine , Pommey Sandra , Stagg John
Published: July 2016

Signals mediated by the C-X-C chemokine ligand 12 (CXCL12; stromal-derived factor 1) and its receptor CXCR4 have been implicated in cancer progression through multiple tumorigenesis processes, including cancer cell proliferation, survival, invasion and tumor angiogenesis. In vitro and murine models recently demonstrated that this axis is also involved in the mechanism of tumor immune evasion and resistance to immune checkpoint inhibitors. In human ovarian cancer, this axis has been associated with ascites, nodes and peritoneal metastasis.

We investigated the prognostic value of CXCL12 protein expression and association with CD8+ T cell infiltration in a cohort of 203 patients diagnosed with a high-grade serous ovarian carcinoma (HGSC). Quantitative immunofluorescence allowed us to evaluate the frequency and level of expression of CXCL12 and CD8+ cell density in the epithelial and stromal tumor compartments. CXCL12 expression in the epithelial tumor compartment was significantly higher than in the stromal compartment (p<0.0001). Strikingly, high levels of tumoral CXCL12 expression were significantly associated with a worse progression-free survival (PFS) and overall survival (OS) (log-rank test, p = 0.007 & p = 0.006, respectively). The Kaplan-Meier median PFS and OS were respectively of 17±1.5 and 42±6.9 months in patients with high levels and of 18±3.6 and 61±7.5 months in patients with low levels of CXCL12. Notably, multivariate analysis showed that CXCL12 levels were associated with a worse outcome independent of FIGO stage and residual disease. No correlation was observed between CXCL12 expression levels and CD8+ T cell density. Intriguingly, in CD8+ subgroup analyses, the prognostic value of CXCL12 levels was restricted to tumors with low intraepithelial CD8+ T cell densities.

Flow cytometry and quantitative real-time PCR results suggest that CXCL12 promotes a mesenchymal phenotype and increased expression of cancer stem cells markers on human ovarian cancer cells.

In conclusion, modulation of the CXCL12/CXCR4 axis is a promising approach in HGSC that may potentiate immunotherapies and may act on epithelial-to-mesenchymal transition. Our study highlights the rationale to evaluate in the clinical setting treatments targeting the CXCL12/CXCR4 in HGSC, specifically in patients lacking CD8+ T cell tumor infiltration.

Citation Format: Laurence Buisseret, Vincent Delisle, Laudine Communal, Sandra Pommey, John Stagg. Prognostic significance of CXCL12 protein levels in high-grade serous ovarian 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 443.
Abstract 4430: Implication of the primary cilium in the Hedgehog pathway in colorectal cancer cells
Authors: Senicourt Blanche
Published: July 2016

Background: The Hedgehog (HH) pathway is involved in the maintenance of numerous cell types both during development and in the adult. Often deregulated in cancers, its involvement in colorectal cancer has come into view during the last few years, although its role remains poorly defined. In most tissues, the HH pathway is highly connected to the primary cilium (PC), an organelle not expressed in the normal colonic epithelium which recruits the functional components and regulates the pathway.

Aims: Since the intestinal epithelium is known to be a non-ciliated tissue, the HH pathway as related to the PC has not been explored. We investigated the presence of PC in colon cancer tumors and cell lines. Our hypothesis is that PC formation controls the HH activation that has been reported in numerous colon cancer studies.

Materials and methods: We used cellular models to look at HH activation, focusing on the final effector Gli1. The link between PC and the HH pathway was shown by the recruitment of the Smo receptor in the PC. We looked for PC in a subset of 63 tumors from a Tissue Micro Array and in 4 colorectal cell lines by immunofluorescence using two well-known markers, acetylated α-tubulin and polyglutamylated tubulin. 3D deconvoluted pictures were obtained to characterize the shape and size of PC. Using cellular models we investigated HH pathway expression by qPCR. We assessed the functional link between HH pathway and PC through localization of the Smo receptor in the PC using immunofluorescence.

Results: We observed the presence of the PC in the epithelium of primary colorectal tumors at all stages but not in their normal counterparts. Using human colorectal cancer cell lines we found a clear correlation between the presence of the PC and the expression of the final HH effector, GLI1, and provide evidence of a functional link between the two by demonstrating the recruitment of the SMO receptor to the PC membrane.

Conclusion: We conclude that the PC directly participates in the HH pathway in colorectal cancer cells which is the first observation of PC expression in tumors arising from a non-ciliated tissue. Functional studies are required to determine the mode of action of this organelle in carcinogenesis.

Citation Format: Blanche Senicourt. Implication of the primary cilium in the Hedgehog pathway in colorectal cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4430.
Abstract 4432: Targeted TET oxidase activity through methyl-CpG binding domain extensively suppresses cancer cell proliferation
Authors: Fukushige Shinichi , Mizuguchi Yasuhiko , Chakma Kanchan , Saiki Yuriko , Horii Akira
Published: July 2016

DNA demethylating agents are useful therapeutic drugs for human cancer; 5-aza-2’-deoxycytidine and 5-azacytidine for myelodysplastic syndrome are good examples. They not only induce DNA demethylation but also have significant cytostatic and cytotoxic effects. However, precise mechanisms for anticancer activity of these demethylating reagents have yet to be established, mainly due to the lack of method to induce only DNA demethylation. Here we show that a fusion protein comprising the methyl-CpG binding domain (MBD) and the catalytic domain of Ten-eleven translocation protein 1 (TET1-CD) globally demethylates and upregulates a number of methylated genes. Gene expression microarray analyses using human embryonic kidney cell line 293T indicate that cells expressing wild-type (wt) TET1 catalytic domain with MBD (MBD-TET1-CDwt) upregulates more genes than ones expressing TET1-CDwt without MBD (TET1-CDwt) or catalytically inactive TET1-CD mutant (mut) with MBD (MBD-TET1-CDmut) and their upregulated genes frequently contained CpG islands (CGIs) within ± 1,000-bp of the transcription start site (TSS). Interestingly, 65% of genes upregulated 5-fold or more by MBD-TET1-CDwt were also reactivated after treatment with 5-azacytidine, DNA demethylating agent. These results suggested a fact that gene reactivation by both methods was primarily based on DNA demethylation. In order to examine growth inhibitory effects of DNA demethylation to cancer cells, we utilized a tetracycline inducible system to regulate the expression of MBD-TET1-CDwt using a prostate cancer cell line. Induction of MBD-TET1-CDwt demethylated and upregulated the glutathione S-transferase pi 1 (GSTP1), one of the hypermethylated genes in prostate cancer. In accordance with reactivation of methylated genes, induction of MBD-TET1-CDwt extensively suppressed the growth of LNCaP cells. Flow cytometry analysis showed decreased cells in S phase and increased cells in G1 phase by MBD-TET1-CDwt expression. Our present results clearly indicate that TET oxidase activity recruited at methyl-CpG sites through MBD induces reactivation of hypermethylated genes by DNA demethylation, and that our methods allow us to analyze the effects of global DNA demethylation in a wide variety of cancer cells.

Citation Format: Shinichi Fukushige, Yasuhiko Mizuguchi, Kanchan Chakma, Yuriko Saiki, Akira Horii. Targeted TET oxidase activity through methyl-CpG binding domain extensively suppresses cancer cell proliferation. [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 4432.
Abstract 444: Hyaluronan-mediated motility receptor (RHAMM) in ovarian cancer
Authors: Buttermore Stephanie , Kruk Patricia
Published: July 2016

This study focuses on Hyaluronan Mediated Motility Receptor (RHAMM) protein expression in ovarian carcinoma (OC). RHAMM protein increases migration, invasion and is associated with poor prognosis when overexpressed. It is a dualistic oncogenic protein located within and on the cell surface acting as a receptor for Hyaluronan (HA). RHAMM is not generally overexpressed in normal tissues, but is upregulated in a number of cancers and is associated with poor clinical outcome. However, the role of RHAMM in OC progression is unknown.

Hypothesis: Elevated RHAMM levels promote OC progression and represent a novel urinary biomarker for OC.

Procedures: Measuring RHAMM expression in OC cell protein lysate and conditioned media (CM) was done by western blot (WB) and densitometry. With institution approval, patient cohort urine from male and female healthy controls and patients with cancer of the ovary, lung, breast, prostate, cervix, colorectum, skin, endometrium, head and neck, and brain were acquired from the H. Lee Moffit Cancer Center at the University of South Florida. Enzyme-linked immunosorbent assay (ELISA) designed for RHAMM detection, was used to measure patient urine expression levels.

Results: Cultured OC cells overexpress and secrete RHAMM. Using OC, SV-40 large T-antigen transfected human ovarian surface epithelial (IOSE) cells (HIO118, IOSE-121) and human dermal fibroblasts (HDF), we found >2x the expression of RHAMM protein in BC (MCF7) and OC (OVCAR5, OV2008, C13) cells by WB. WB of CM indicated >3x RHAMM levels secreted by OC compared to IOSE cells.

Urinary levels of RHAMM are elevated in OC patients. Since RHAMM is not an integral membrane protein, but rather loosely tethered to the plasma membrane, we determined whether RHAMM could be detected in bodily fluids of OC patients. ELISA showed detection of urinary RHAMM protein levels averaged 0.140 ng/ml (n = 26) in healthy patient controls whereas OC patients averaged 0.411 ng/ml (n = 132).

Conclusions: Prior to this study, overexpression and secretion of RHAMM by OC cells had not been reported. This has expanded our knowledge of OC etiology by delineating a functional role for RHAMM-driven OC progression. Validating elevated urinary levels of RHAMM suggests it is a potential diagnostic marker alone or in combination with other markers for OC detection. Further, the ability to monitor OC through the course of disease using urinary RHAMM levels could indicate recurrent disease or therapeutic efficacy. Understanding RHAMM expression in OC may lead to a possible diagnostic/prognostic indicator or therapeutic target for OC.

Citation Format: Stephanie Buttermore, Patricia Kruk. Hyaluronan-mediated motility receptor (RHAMM) in ovarian 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 444.
Abstract 4441: Non-pathogenic bacteria change host DNA methylation in vivo
Authors: Sun Ang , Jelinek Jaroslav , Maegawa Shinji , Jobin Christian , Issa Jean-Pierre
Published: July 2016

Introduction: More than a thousand species of bacteria live in a normal person's gastrointestinal tract. Epigenetic effects on the colon directly caused by the commensal bacteria remain elusive. In the current study, we explore the bacterial effects on DNA methylation of the host colon. Methods: Eight non-pathogenic bacterial species were introduced to the gut of germ-free (GF) mice to make specific-pathogen free (SPF) mice. Our study consisted of the following groups (n = 6 for each): wildtype (WT) GF, WT SPF, IL-10 knockout (KO) GF, IL-10 KO SPF, and IL-10 KO GF or SPF treated with azoxymethane (AOM). We analyzed DNA methylation in genomic DNA extracted from the proximal colon using Digital Restriction Enzyme Analysis of Methylation (DREAM) and compared methylation levels at CpG sites among different groups. Results: 1) Principal component analysis (PCA) performed on methylomes of all the samples separated the mouse samples based on presence or absence of bacteria, IL-10 KO status or AOM treatment into 6 distinct groups. 2) We analyzed CpG sites differentially methylated between GF and SPF mice at p<0.05 significance level. In WT mice, there were 1871 CpG sites (11% out of 17475 analyzed sites), in IL10 KO mice, there were 5986 CpG sites (33% out of 18254 analyzed sites) and in IL10 KO AOM-treated mice there were 6755 CpG sites (31% out of 21697 analyzed sites) differentially methylated. 3) There were 756 common CpG sites differentially methylated in all SPF vs GF comparisons. Interestingly, these sites were enriched 2.6-fold for aging-related CpG sites. 4) WT SPF mice showed increased methylation at CpG sites hypomethylated in WT GF mice and, conversely, decreased methylation at CpG sites hypermethylated in WT GF mice. This pattern of DNA methylation change was also observed by comparing GF with SPF in IL-10 KO mice with or without AOM treatment. 5) Both in GF and SPF mice the DNA methylation changes were more pronounced in IL10 KO while AOM had little additional effect. 6) IL10 KO resulted in genome-wide increase of methylation. However, the introduction of bacteria in SPF mice resulted in the methylation change mainly at CpG islands. Conclusions: Non-pathogenic bacteria introduced in the colon cause methylation gains at CpG sites with low DNA methylation and methylation losses at sites with high DNA methylation in a pattern similar to the changes observed in aging and cancer.

Citation Format: Ang Sun, Jaroslav Jelinek, Shinji Maegawa, Christian Jobin, Jean-Pierre Issa. Non-pathogenic bacteria change host DNA methylation in vivo. [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 4441.
Abstract 4447: EZH2 contributes to breast cancer metastasis
Authors: Yomtoubian Shira , Ryu Seongho , Lee Sharrell , Havel Lauren , Gao Dingcheng , Mittal Vivek
Published: July 2016

While the 5-year relative survival rate of localized stage breast cancer is 99%, once the cancer spreads to distant lymph nodes and organs the survival rate falls to 25%. In order to develop strategies to combat metastasis, we have been studying epigenetic factors that contribute to the aggressive nature of breast cancer. We have focused on enhancer of zeste homolog 2 (EZH2), a histone methyl transferase, because breast cancer patients expressing increased EZH2 exhibit highest metastatic recurrence. We have found through in vivo studies that blocking EZH2 activity not only prevents metastasis formation, but also targets established metastases. Genome wide ChIP-seq analysis revealed that EZH2 may impact breast cancer metastasis through regulating the expression of EMT marker, E-cadherin, and stem cell markers, GATA3 and DKK2. Mammosphere assays from tumors treated with pharmacological blockade of EZH2 and limiting dilution analysis validates that EZH2 inhibition impacts the tumor-initiating cell population. Our data suggests that inhibition of EZH2 activity may have anti-metastatic therapeutic potential and may complement current standards of breast cancer therapy.

Citation Format: Shira Yomtoubian, Seongho Ryu, Sharrell Lee, Lauren Havel, Dingcheng Gao, Vivek Mittal. EZH2 contributes to breast cancer metastasis. [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 4447.
Abstract 4448: Development of DNA methylation biomarkers for pancreatic cancer diagnosis
Authors: Shinjo Keiko , Katsushima Keisuke , Nagae Genta , Aburatani Hiroyuki , Yamao Kenji , Kondo Yutaka
Published: July 2016

Pancreatic cancer is highly lethal malignancy despite the recent clinical advancement. Since pancreatic cancer is often difficult to diagnose, there is an urgent need to develop minimally invasive biomarkers for detection. Epigenetic alterations have emerged as a common hallmark of many types of human cancers as well as pancreatic cancer. The presence of cell-free DNA (cfDNA) in blood stream has been known and cfDNA possesses tumor-related genetic alterations as well as epigenetic alterations. Therefore, invention of blood-based DNA methylation biomarkers and their high-sensitive detection system might be a promising diagnostic tool for pancreatic cancer. In order to establish the diagnostic biomarkers, first, we performed genome-wide DNA methylation analysis of 38 EUS-FNA specimens of pancreatic cancer with clinical stage III-IV using Illumina Infinium HumanMethylation450 BeadChip. We further analyzed the DNA methylation profiles of other types of tumors in the public database, TCGA (The Cancer Genome Atlas), and identified five marker genes, which were specifically methylated in pancreatic cancers. In addition, analysis of DNA methylation status of pancreas cancers comparing 10 normal tissues in TCGA data set revealed that 99% of tumors were methylated at least one of the five markers (sensitivity, 98.9%; specificity, 44%). We validated these methylation markers in different FNA sample set (n = 19) by bisulfite pyrosequencing or quantitative methylation specific PCR and found that 18/19 (95%) cases were methylated in at least one marker (AUC = 0.95). Finally, we performed methylation analysis in cfDNA from pancreatic cancer patients. From our preliminary data, we could detect aberrant DNA methylation in cfDNA from pancreatic cancer patients.

We identified potential DNA methylation biomarkers for the diagnosis of pancreatic cancers and these markers might be applicable for detecting cancer in cfDNA.

Citation Format: Keiko Shinjo, Keisuke Katsushima, Genta Nagae, Hiroyuki Aburatani, Kenji Yamao, Yutaka Kondo. Development of DNA methylation biomarkers for pancreatic cancer diagnosis. [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 4448.
Abstract 445: Ultra-deep sequencing detects ovarian cancer cells in peritoneal fluid and reveals somatic TP53 mutations in noncancerous tissues
Authors: Krimmel Jeffrey D. , Schmitt Michael W. , Kennedy Scott R. , Harrell Maribel I. , Agnew Kathy J. , Loeb Larry A. , Swisher Elizabeth M. , Risques Rosa Ana
Published: July 2016

The detection of subclonal tumor-specific somatic mutations in clinical samples could revolutionize cancer diagnostics, but is limited by insufficiently sensitive sequencing methods. Duplex Sequencing is a novel next-generation sequencing (NGS) technology that implements single-molecule barcoding of both strands of DNA to allow internal error correction. This modification reduces the error rate of NGS from 1 in 1000 to less than 1 in 10 million nucleotides, an unprecedented sensitivity that enables accurate ultra-deep sequencing for clinical applications. In this study we sought to determine whether Duplex Sequencing could detect extremely rare TP53 mutated cancer cells disseminated into the peritoneal cavity of women with high-grade serous ovarian carcinoma (HGSOC). HGSOC is the most common and most aggressive type of ovarian cancer, shows early transperitoneal dissemination, and is characterized by near-universal prevalence of driver TP53 mutations. We analyzed 17 peritoneal fluid samples from women with HGSOC and known TP53 tumor mutation (cases) and 20 peritoneal fluid samples from women without cancer (controls). The tumor-specific TP53 mutation was detected in matched peritoneal fluid from 94% of cases (16/17), including 2 patients with occult tubal intraepithelial neoplasia, 7 patients with early stage HGSOC, and 8 with negative peritoneal fluid cytopathology. Tumor-specific alleles were detected as low as <1/25,000. Surprisingly, we also detected additional extremely low-frequency somatic TP53 mutations in peritoneal fluid from nearly all (35/37) cases and controls. These mutations were more abundant in cases than in controls, and in the latter correlated with age. The total burden of somatic TP53 mutations (tumor specific and non-specific) in a peritoneal fluid sample could distinguish cases from controls with 82% sensitivity (14/17) and 90% specificity (18/20). Duplex Sequencing also revealed low-frequency, age-associated somatic TP53 mutations in 100% (15/15) of matched peripheral blood samples. Our results demonstrate the ability of Duplex Sequencing to detect very rare cancer cells, but also provide evidence of widespread, low frequency somatic TP53 mutation in non-cancerous tissue. This compromises the specificity of TP53 mutation tests for cancer detection in liquid biopsies.

Citation Format: Jeffrey D. Krimmel, Michael W. Schmitt, Scott R. Kennedy, Maribel I. Harrell, Kathy J. Agnew, Larry A. Loeb, Elizabeth M. Swisher, Rosa Ana Risques. Ultra-deep sequencing detects ovarian cancer cells in peritoneal fluid and reveals somatic TP53 mutations in noncancerous tissues. [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 445.
Abstract 4452: Oxdative stress suppresses the expression of 15-hydroxyprostaglandin dehydrogenase via upregulation of DNMT3a/Snail in human colon epithelial cells
Authors: Lee Ja-Young , Lee Jeong-Eun , Na Hye Kyung
Published: July 2016

Overproduction of prostaglandin E2 (PGE2) is implicated in pathogenesis of inflammation-associated carcinogenesis. 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is a key enzyme that catalyzes the conversion of PGE2 to biologically inactive 15-keto-PGE2 metabolite. 15-PGDH has been considered to be a tumor suppressor as its expression is frequently repressed in human malignancies via hypermethylation of its promoter. In our previous study, oxidative stress caused by Helicobacter pylori infection suppressed the expression of 15-PGDH. Reactive oxygen species (ROS) can induce methylation as well as mutation of genes including those that encode tumor suppressor proteins, thereby contributing to carcinogenesis. This prompted us to investigate whether 15-PGDH expression could be down-regulated by oxidative stress in normal colon epithelial CCD841 cells. When CCD841 cells were treated with H2O2, the expression level of 15-PGDH was significantly reduced in both concentration and time dependent manners. Hydrogen peroxide (300 μM) induced the expression of DNA methyltransferase (DNMT) 3a, which was blocked by an inhibitor of DNMT, 5-Aza-2′-deoxycytidine (5-Aza). The 5-Aza abrogated the down-regulation of 15-PGDH expression induced by H2O2. The antioxidant, N-acetylcysteine (NAC) abrogated the down-regulation of 15-PGDH and upregulation of DNMT 3a expression induced by H2O2. The transcriptional repressor Snail plays a central role in the suppression of 15-PGDH expression. H2O2 treatment also elevated expression of Snail, which was abrogated by NAC and 5-Aza. Taken together, these finding suggest that ROS suppresses the expression of 15-PGDH through hypermethylation of CpG island in the 15-PGDH promoter.

Citation Format: Ja-Young Lee, Jeong-Eun Lee, Hye Kyung Na. Oxdative stress suppresses the expression of 15-hydroxyprostaglandin dehydrogenase via upregulation of DNMT3a/Snail in human colon 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 4452.
Abstract 4475: SMYD2 inhibitor BAY-598 reveals the giant scaffold AHNAK as a novel multi-methylated substrate
Authors: Eggert Erik , Stellfeld Timo , Korr Daniel , Stresemann Carlo
Published: July 2016

The SET and MYND domain-containing protein 2 (SMYD2) has been reported to mono-methylate several lysine residues on histone and non-histone proteins. Overexpression of SMYD2 has been reported in cancer cell lines as well as in esophageal squamous cell carcinoma (ESCC), bladder carcinoma, gastric cancer and pediatric acute lymphoblastic leukemia patients and correlates with lower survival rates. Although several studies uncovered important roles of SMYD2 in promoting tumorgenesis by protein methylation of key proteins (e.g. p53, PTEN, Rb, PARP), the biology of SMYD2 and its possible role in cancer biology is still far from being fully understood. Therefore, we employed the SMYD2 inhibitor BAY-598 (AACR-2015 abstract 2829, publication in preparation), and used proteomics to find novel substrates of SMYD2 in cancer cells.

We confirmed with both BAY-598 and by knockdown experiments that the giant scaffold protein AHNAK is a novel major substrate of SMYD2. Different studies have tried to decipher the function of AHNAK in normal and diseased tissues. AHNAK forms multi-protein complexes acting as a structural scaffold involved in different processes, including calcium channel regulation, cell contact signaling, migration, and tumor metastasis. Interestingly, we found AHNAK to be mono-methylated by SMYD2 on multiple sites in its central repeated domain as well as on its C-terminal domain. BAY-598 potently inhibited methylation at these sites on AHNAK. To further determine the prevalence of AHNAK methylation, we analyzed different cancer cell lines and tissues from mice. AHNAK methylation correlated with SMYD2 expression, but not with AHNAK expression, suggesting tissue-specific regulation of AHNAK methylation by SMYD2.

In summary, the novel finding of AHNAK regulation by methylation further complements the understanding of the roles of SMYD2 in cancer biology and underlines the utility of using probe inhibitors like BAY-598 to explore potential therapeutic targets in cancer.

Citation Format: Erik Eggert, Timo Stellfeld, Daniel Korr, Carlo Stresemann. SMYD2 inhibitor BAY-598 reveals the giant scaffold AHNAK as a novel multi-methylated substrate. [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 4475.