bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5494: Hypoxia via HIF1alpha can regulate Wnt signaling in human colon cancer cells
Authors: Lyou Yung , Habowski Amber , Sprowl-Tanio Stephanie , Pate Kira , Chen George , Waterman Marian L.
Published: July 2017

Standard-of-care treatment for metastatic colorectal cancer combines chemotherapy with bevacizumab, an angiogenesis inhibitor that depletes nutrients and triggers hypoxia. Unfortunately, this strategy extends patient survival only a few months because tumors acquire adaptive resistance, enabling resumption of angiogenesis. Adaptive resistance arises from reprogrammed metabolism, but the regulatory networks that govern reprogramming are not defined. Our studies have shown that overactive Wnt signaling, which is the most common cause of colon cancer, programs cancer metabolism by promoting glycolysis and angiogenesis. Interestingly, metabolic genes targeted by Wnt are also regulated by the hypoxia transcription factor HIF1alpha suggesting there may be crosstalk between the two pathways. Several studies suggest that HIF1alpha can influence Wnt signaling, but the reported effects are variable ranging from positive to negative. We find that HIF1alpha and LEF/TCF/beta-catenin complexes co-regulate metabolic targets including LEF1 and TCF1 (TCF7) expression. Using luciferase reporters driven by LEF/TCF promoters, we observe significantly increased LEF1/TCF1 but not TCF3(TCF7L1)/TCF4(TCF7L2) promoter activity in hypoxic colon cancer cells. We identified putative hypoxia responsive elements (HREs) in the human LEF1 promoter and deletion of these HREs reduced responsiveness to hypoxia. Furthermore, when colon cancer cells were treated with inhibitors that either inactivate or reduce HIF1alpha protein, there was a significant decrease in Wnt reporter activity. These results suggest that hypoxia and the Wnt pathway crosstalk wherein hypoxia co-regulates metabolism genes and increases Wnt signaling capacity via LEF/TCF expression. Current studies are underway to investigate the mechanisms of crosstalk and joint regulation of metabolic gene programs during adaptive resistance.

Citation Format: Yung Lyou, Amber Habowski, Stephanie Sprowl-Tanio, Kira Pate, George Chen, Marian L. Waterman. Hypoxia via HIF1alpha can regulate Wnt signaling in human colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5494. doi:10.1158/1538-7445.AM2017-5494
Abstract 550: Predictive value of co-expression network consisting of FOXM1 and its neighbor genes in cancers
Authors: Song Bic-Na , Chu In-Sun
Published: July 2017

Purpose: Forkhead box protein M1 (FOXM1) is a key regulator of tumorigenesis. Previous studies demonstrated that FOXM1 expression was strongly correlated with poor prognosis of various cancers. The aim of this study is to define a signature consisting of FOXM1 and its associated genes and assess its prognostic potential in cancers including hepatocellular carcinoma (HCC) and others.

Experimental procedures: We analyzed a gene expression profile of 100 patients with HCC to identify FOXM1-correlated genes (the FOXM1 signature) associated with the prognosis of HCC patients. The validity of the FOXM1 signature was verified in an independent HCC cohort (n = 242). Using the signature, we also analyzed different cancer types including pancreatic adenocarcinoma, lung adenocarcinoma, breast carcinoma, and bladder urothelial carcinoma to verify the association between the FOXM1 signature and prognosis. Various statistical methods were applied to signature finding and validation. Upstream regulator and gene-to-gene network analyses were performed to identify potential key mediators of the FOXM1 signature.

Results: Through a gene expression profiling in 100 patients with HCC, we identified a gene set consisting of FOXM1 and its co-expressed genes for predicting poor prognosis. A predictive value of the signature was validated in an independent cohort containing 242 HCC patients. In multivariate analysis, the FOXM1 signature showed the most significant prognostic value (HR = 1.706, 95% CI = 1.176-2.475, P = 0.005). By directly applying the signature to four data sets in different cancer types, our signature showed a consistent prognostic significance, regardless of tumor types. Finally, upstream regulator and gene-to-gene network analyses found an interconnection of network hubs composed by FOXM1, MYC, and E2F1 that might be common key mediators of patient survival in cancer.

Conclusions: The FOXM1 signature represents a promising common diagnostic tool to identify high-risk cancer patients and may extend treatment options in various cancers.

Note: This abstract was not presented at the meeting.

Citation Format: Bic-Na Song, In-Sun Chu. Predictive value of co-expression network consisting of FOXM1 and its neighbor genes in cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 550. doi:10.1158/1538-7445.AM2017-550
Abstract 5502: Functional evaluation of superenhancers as mediators of epithelial ovarian cancer risk
Authors: Vavra Kevin C. , Coetzee Simon , Lee Janet M. , Pharoah Paul , Hazelett Dennis J. , Lawrenson Kate , Gayther Simon A.
Published: July 2017

Superenhancers are master regulators of genes involved in cellular differentiation and tumorigenesis. While it is now well established that typical enhancers are key mediators of alleles associated with a mild risk of polygenic diseases, including cancer, the role of superenhancers is less understood. We evaluated the role of superenhancers in mediating risk of epithelial ovarian cancer (EOC). Using FunciVar software and superenhancers defined using H3K27ac chromatin immunoprecipitation followed by next generation sequencing, performed in cell lines and tissues, we characterized the superenhancer-risk SNP intersect and identified a series of risk-associated loci where multiple candidate causal alleles overlap with ovarian superenhancers. In particular, locus 3q25, the locus most associated with epithelial ovarian cancer (EOC) risk from GWAS studies, has >70 candidate causal risk SNPs, all of which coincide with superenhancers detected in ovarian cancer precursor cells (ovarian and fallopian tube epithelia) but not in ovarian cancers. We are proposing that the presence of one or more SNPs occurring at this locus leads to a higher expression of one or more genes by modulating activation of the superenhancer region. We tested the effect of decreasing the binding affinity of BRD4, a protein that binds superenhancer regions, using a small molecule inhibitor on the expression of genes in this locus. We identified three candidate genes—LEKR1, SSR3, and TiPARP—that demonstrated a decrease expression level as a result of BRD4 binding inhibition.

Preliminary results show that partial knockdown of either SSR3 or TiPARP decreases normal ovarian and fallopian cell proliferation, and knocking down both SSR3 and TiPARP simultaneously shows an enhanced proliferation decrease compared with knockdown of a single gene. We then created stable knockout (KO) models using the CRISPR-Cas9 system to fully KO one, two or all three of our candidate causal genes: LEKR1, SSR3, and TiPARP. Functional evaluation of these KO models is currently underway to test the hypothesis that risk SNPs may regulate more than one gene at this locus. In all we identify a potential role for superenhancers at a subset of EOC risk loci, and present a functional pipeline for identification of the target gene/genes and evaluation of their role in neoplastic transformation.

Citation Format: Kevin C. Vavra, Simon Coetzee, Janet M. Lee, Paul Pharoah, Dennis J. Hazelett, Kate Lawrenson, Simon A. Gayther. Functional evaluation of superenhancers as mediators of epithelial ovarian cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5502. doi:10.1158/1538-7445.AM2017-5502
Abstract 5508: Histone modifications affect differential regulation of TGF-beta-induced Nox4 by p53
Authors: Boudreau Howard E. , MA Wei Feng , Korzeniowska Agnieszka , Park Jonathan J. , Leto Thomas L.
Published: July 2017

Recent studies suggest p53 plays an important role in TGF-β-mediated cell signaling and migration. Previously, we showed wild-type (WT) and mutant (mt) forms of p53 differentially regulate reactive oxygen species (ROS) generation by NADPH oxidase-4 (NOX4). We found that WT-p53 suppresses TGF-β-induced NOX4, ROS production, and cell migration, whereas tumor-associated p53-mt proteins enhance NOX4 expression and cell migration by TGF-β/SMAD3-dependent mechanisms. In this study, we utilized The Cancer Genome Atlas (TCGA) to perform statistical analysis on gene expression data from primary tumor samples and found a correlation between tumors with p53 “hotspot” mutations and increased NOX4 mRNA expression. Furthermore, we examined the basis of human NOX4 promoter regulation by p53 and SMAD3. By deletion analysis of the NOX4 promoter, we found two critical SMAD3 binding elements (SBE) are also required for p53-dependent promoter activity. Conversely, promoter activity was abolished by dose-dependent heterologous expression of p53-WT. Moreover, expression of active SMAD3 resulted in robust NOX4 promoter activity, which was abolished when co-expressed with p53-WT. Chromatin immunoprecipitation (ChIP) analysis revealed SMAD3 and p53-WT or p53-mt were associated with specific SBEs and p53 response elements (p53-RE) in a TGF-β-dependent manner. Interestingly, the repressive effect by p53-

WT on NOX4 was relieved upon treatment with histone deacetylase (HDAC) inhibitors or mutation of the transactivation domain. Overexpression of p300, a known p53-mt-binding transcriptional co-regulator and histone acetyltranserfase (HAT), enhanced p53-mt-mediated NOX4 promoter activity, whereas the HAT-inactive p300-mt reduced promoter activity. Furthermore, overexpression of p53-mt augmented TGF-β-mediated acetylation of histones associated with the NOX4 promoter. Finally, scratch wound assays demonstrated NOX4 and p300 promote TGF-β/mutant p53-mediated cell migration. Collectively, these data and provide new insight on TGF-β/SMAD3 and mutant p53-mediated NOX4 induction involving epigenetic control of NOX4 in tumor cell migration.

Citation Format: Howard E. Boudreau, Wei Feng MA, Agnieszka Korzeniowska, Jonathan J. Park, Thomas L. Leto. Histone modifications affect differential regulation of TGF-beta-induced Nox4 by p53 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5508. doi:10.1158/1538-7445.AM2017-5508
Abstract 5511: Lineage-specific and shared cytokine-sensing genes respond distinctly to the master regulator STAT5
Authors: zeng xianke , Willi Michaela , Shin Ha Youn , Wang Chaochen , Hennighausen Lothar
Published: July 2017

JAK/STAT signaling pathways are critical for cell response to cytokines across multiple tissues, genetic variation in this pathway could associate with neoplastic diseases. STAT5, the most common used STAT family member, differently activates two distinct classes of genes. It activates cell-specific genes up to two orders of magnitude higher than shared genes expressed across different cell types. Socs2 (Suppressors of cytokine signaling 2), a widely expressed STAT5 target gene and a negative regulator of cytokine feedback loops, contains STAT5 response element within its promoter region. In our study, we demonstrated that STAT5 preferentially binds to promoter sequence of shared STAT5 targeted genes and to distal putative enhancers of lineage-specific genes. DNase-seq demonstrated that chromatin accessibility of STAT5-based enhancers was greatly dependent on cytokine exposure, while common STAT5 promoter targets were constitutively accessible. CRIPSR /Cas9 gene editing was used to delete the generic STAT5 binding sites in Socs2 locus in mice. Prolactin induction of Socs2 was abrogated both in liver and mammary gland, and resulted in elevated STAT5 activation and precocious mammary development in mutant mice. Taken together, we demonstrated that promoter-bound STAT5 modulates cytokine responses of universal STAT5 target genes, while enhancer-bound STAT5 is necessary for linage-specific target gene activation. This study provides new insight into the mechanism of used by a generic cytokine response element controlling lineage-specific transcription.

Citation Format: xianke zeng, Michaela Willi, Ha Youn Shin, Chaochen Wang, Lothar Hennighausen. Lineage-specific and shared cytokine-sensing genes respond distinctly to the master regulator STAT5 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5511. doi:10.1158/1538-7445.AM2017-5511
Abstract 5518: Rgs8 and Rgs16 are tumor suppressor genes in mouse pancreatic ductal adenocarcinoma
Authors: Choudhuri Shreoshi Pal , Zolghadri Yalda , Mascarenhas Luke , Ocal Ozhan , Wilkie Thomas
Published: July 2017

We have identified regulators of G protein signaling (Rgs8 and Rgs16) as a new class of tumor suppressor genes in a mouse model of pancreatic ductal adenocarcinoma (PDA). PDA is the 3rd leading cause of cancer related deaths in the United States. Kras mutations (e.g. KrasG12D) are associated with over 90% of human PDA and are an early event in the multistep process leading to PDA. Kras can be activated by protein kinase and G-Protein Coupled Receptor (GPCR) signaling. Rgs proteins regulate GPCR signaling by accelerating the GTPase activity of Gq- and Gi class alpha subunits. Activating alleles of Gq that are resistant to Rgs inhibition are associated with PDA in humans. We found Rgs8 and Rgs16 are in vivo reporters of Kras activity in pancreatic intraepithelial neoplasia (PanIN), intraductal papillary mucinous neoplasm (IPMN), and PDA progression (DMM 8, 2015). Rgs8 and Rgs16 are expressed in PanIN and IPMN, precursors of PDA, in KC mice (LSL-KrasG12D; p48::Cre). To test if Rgs8-16 function as tumor suppressor genes, we crossed the Rgs8-16 double knockout into KC (termed KCR8-16) mice. Compared to KC, PDA initiates earlier, is more aggressive, and KCR8-16 mice die earlier. Our study suggests that Rgs8 and Rgs16 control Kras-dependent PDA initiation and progression.

Note: This abstract was not presented at the meeting.

Citation Format: Shreoshi Pal Choudhuri, Yalda Zolghadri, Luke Mascarenhas, Ozhan Ocal, Thomas Wilkie. Rgs8 and Rgs16 are tumor suppressor genes in mouse pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5518. doi:10.1158/1538-7445.AM2017-5518
Abstract 5520: CCN5/WISP-2 is a negative regulator of epithelial to mesenchymal transition and stemness in breast cancer
Authors: Maity Gargi , Das Amlan , Sarkar Sandipto , Banerjee Snigdha , Banerjee Sushanta K.
Published: July 2017

Background and Objective: Breast cancer is the most common cancer in women and a leading cause of cancer mortality in western countries. CCN5 (also known as Wnt-1-induced signaling protein-2 or WISP-2) is a 29-31-kDa matricellular protein that plays as a negative regulator of breast carcinoma. Our previous studies had shown the importance of CCN5/ WISP-2 in the suppression of breast and pancreatic cancer progression through the regulation of the invasive phenotypes. Considering the previous report, our aim is to investigate whether human recombinant CCN5 inhibit pathobiological events like epithelial to mesenchymal transition (EMT), migration and stemness in triple negative breast cancer cells.

Methods: To investigate the negative impact of CCN5 on EMT and stemness of TNBC, we performed several techniques like western blot, clonoogenic assay, soft agar assay, sphere formation assay etc.

Results and Conclusions: The exposure of triple negative human breast cancer cells (TNBC), MDA-MB-231 and HCC-70, to recombinant CCN5 (hrCCN5), resulted in a dose-dependent inhibition of cell-proliferation through the induction of apoptotic cell death. The treatment of hrCCN5 regulates various pathobiological events in breast cancer cells, such as reprogramming the mesenchymal to epithelial transition (MET) followed by reduction of stemness features as confirmed by sphere formation assay and delaying in vitro migration. Finally, treatment with hrCCN5 in TNBC cells significantly inhibited anchorage-dependent and independent growth of TNBC. Collectively, CCN5’s control of cancer cell physiology indicates that hrCCN5 has the potential of being used as a major therapeutic agent against triple negative breast cancer.

Citation Format: Gargi Maity, Amlan Das, Sandipto Sarkar, Snigdha Banerjee, Sushanta K. Banerjee. CCN5/WISP-2 is a negative regulator of epithelial to mesenchymal transition and stemness in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5520. doi:10.1158/1538-7445.AM2017-5520
Abstract 5523: Loss of RAB25 cooperates with oncogenes in the transformation of human mammary epithelial cells
Authors: Rao Krishna , Joshi Pooja Sridhar
Published: July 2017

The RAB guanosine triphosphates (RAS-related in brain) belong to the Ras superfamily of GTPases, and loss of RAB 25 expression has been reported in a number of breast cancer cases containing H-Ras point mutations, particularly triple negative breast cancers (TNBC), one of the most aggressive subtypes of breast cancer and associated with a poor prognosis. The mechanism involved in the progression of these tumors is poorly understood.

In this study, we are trying to understand if loss of RAB25 expression in Human Mammary Epithelial Cell (HMEC) lines co-operates with H-Ras mutations and contributes to tumorigenesis. HMEC were immortalized by transduction with LXSN CDK4 R24C, a mutant form of cyclin-dependent kinase, followed by transduction with hTERT, catalytic subunit of the telomerase enzyme that permits the cells to exceed the Hayflick Limit and become immortal.

We have found that with loss of RAB25 and over expression of mutant H-Ras61L, immortal HMEC undergo transformation. We have looked into the co-operativity between loss of Rab25 and H-Ras61L mutant by in-vitro studies to show their anchorage independent growth and increased ability to migrate. Furthermore, cells express low CD24, high CD44, and very low levels of Claudin indicating that cells acquire stem-like properties upon transformation. Loss of RAB25 and over-expression of H-ras61L resulted in increased expression of transcription markers Snail and Slug that drive these cells to lose E-cadherin and undergo Epithelial Mesenchymal Transition (EMT). We also intend to carry out nude mice studies to look for the potential of these cells to form tumors and metastasize.

This study shows that loss of RAB25 and over-expression of mutant H-Ras can transform HMEC and give rise to mesenchymal stem-like tumors. Our findings reveal that RAB25 functions as a tumor suppressor gene, and loss of RAB25 could serve as a novel biomarker in the prognosis of Claudin-low type of TNBC.

Citation Format: Krishna Rao, Pooja Sridhar Joshi. Loss of RAB25 cooperates with oncogenes in the transformation of human mammary epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5523. doi:10.1158/1538-7445.AM2017-5523
Abstract 5524: Gene 33/Mig6 regulates apoptosis and the DNA damage response through independent mechanisms
Authors: Li Cen , Park Soyoung , Zhang Xiaowen , Eisenberg Leonard , Zhao Hong , Darzynkiewicz Zbigniew , Xu Dazhong
Published: July 2017

Gene 33 (Mig6, ERRFI1) is an inducible adaptor/scaffold protein whose expression can be induced by both stress and mitogenic signals. It contains multiple domains for protein-protein interaction and is involved in a broad spectrum of cellular functions including cell proliferation, cell migration, cell apoptosis, and cell senescence. Many functions of Gene 33 are attributable to its ability to bind to the kinase domain of the EGFR family receptor tyrosine kinases thereby inhibiting their activities. Gene 33 also binds and activates tyrosine kinase c-Abl and is involved in other signing pathways such as NF-κB, HGF, and JNK. Growing evidence at the genetic, cellular, and animal levels indicates that Gene 33 functions as a tumor suppressor in the lung. Gene 33 has been shown to promote apoptosis. A recent study has linked this protein to the DNA damage response induced by hexavalent chromium [Cr(VI)]. In the current study we find that ectopic expression of Gene 33 strongly induces apoptosis by both activating the c-Abl/p73 pathway and inhibiting the EGFR/AKT pathways in BEAS-2B lung epithelial cells and A549 lung carcinoma cells. Surprisingly, ectopic expression of Gene 33 also triggers the DNA damage response in an ATM-dependent fashion and through pathways with and without association with apoptosis. We observed striking presence of Gene 33 in the nucleus and chromatin, which is in contrast with the belief that Gene 33 is an exclusively cytoplasmic protein. Our data show that chromatin localization of Gene 33 is partially dependent on the EBD motif of Gene 33, a domain required for interaction with EGFR and c-Abl. Our data also indicate that Gene 33 may regulate chromatin targeting of c-Abl and EGFR. Furthermore we find that both endogenous and ectopically expressed Gene 33 strongly interact with histone H2AX. Our data have revealed potential nuclear/chromatin-associated mechanisms that underline the function of Gene 33 in apoptosis and the DNA damage response.

Citation Format: Cen Li, Soyoung Park, Xiaowen Zhang, Leonard Eisenberg, Hong Zhao, Zbigniew Darzynkiewicz, Dazhong Xu. Gene 33/Mig6 regulates apoptosis and the DNA damage response through independent mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5524. doi:10.1158/1538-7445.AM2017-5524
Abstract 5526: Tumor suppressive role of BMI-1 through inhibition of JAK-STAT signaling in leukemia
Authors: Lam Yuk Man , Lam Stephen Sze Yuen , Leung Anskar Yu Hung , Ng Ray Kit
Published: July 2017

BMI-1, which is one of the core components of polycomb repressive complex 1, is frequently found deregulated in patients with hematological disorders. In last decades, researchers concordantly agree that BMI-1 mediates tumorigenesis of leukemia stem cells through p16INK4A leukemogenic pathway. However, accumulating evidences contradict the idea that BMI-1 solely plays an oncogenic role in tumorigenesis. It has been shown BMI-1 depletion favors the development of myelofibrosis in mice; whereas high BMI-1 expression suppressed colony forming ability of MLL-ENL-transformed bone marrow and correlated with higher survival in some cancers. In this study, we hypothesized that BMI-1 plays a tumor suppressive role, which is independent of the regulation of INK4A-ARF locus, in human leukemia. BMI-1 was over-expressed in a panel of myeloid and lymphoid lineage leukemia cells, including HL-60, MV4-11, MonoMac-6, SEM, Nalm-20 and RS4;11. We observed no deregulation of p16INK4A and p14ARF genes by BMI-1, suggesting the regulation of INK4A-ARF locus is independent of BMI-1 modulation in leukemia cells. Nevertheless, over-expression of BMI-1 resulted in significant reduction of leukemia cell proliferation. It is noted that constitutively active JAK-STAT signaling pathway is crucial to leukemia cell survival. By modulation of BMI-1 level, we demonstrated suppression of the activated JAK-STAT signaling pathway in most of the leukemia cell lines with the exception of MonoMac-6 and RS4;11. This is in agreement with the high sensitivity to ruxolitinib, a JAK-STAT inhibitor, in all the tested leukemia cell lines except RS4;11. Importantly, we showed that BMI-1 over-expression could sensitize RS4;11 cells to reduce cell proliferation under ruxolitinib treatment. These results suggest that higher efficacy of ruxolitinib treatment could be achieved under a condition of high cellular level of BMI-1. We further demonstrated that ruxolitinib treatment was more effective in a cohort of AML patient samples (n = 25) with a context of higher BMI-1 expression (p < 0.05). Altogether, our results suggest that BMI-1 functions as a tumor suppressor gene via inhibition of the JAK-STAT signaling pathway. The endogenous level of BMI-1 could be served as an indicator for the effective treatment of JAK-STAT-dependent leukemia cells using ruxolitinib.

Citation Format: Yuk Man Lam, Stephen Sze Yuen Lam, Anskar Yu Hung Leung, Ray Kit Ng. Tumor suppressive role of BMI-1 through inhibition of JAK-STAT signaling in leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5526. doi:10.1158/1538-7445.AM2017-5526
Abstract 5527: Effects of PTEN localization and phosphatase activity on gene expression profiling of glioblastoma cells
Authors: Wang Yubing , Chan Andrew M.
Published: July 2017

PTEN possesses both protein and lipid phosphatase activities, with its tumor-suppressor function mainly depends on its lipid phosphatase activity through inhibiting the PI3K/AKT signaling pathway at the cell membrane. However, increasing evidence have demonstrated that PTEN also localizes to other intracellular organelles such as the endoplasmic reticulum, the mitochondria, or the nucleus where it plays important roles in multiple cellular processes. To further understand how PTEN localization affects the gene expression profiling in cancer cells, a PTEN null glioblastoma cell line, U87MG, was stably expressing wild-type, nuclear localization signal (NLS)-tagged, nuclear export signal (NES)-tagged and myristoylation (MYR)-tagged PTEN. Ion Torrent based whole transcriptome sequencing was used for gene expression analysis. Gene ontology enrichment analysis showed that the most common cellular component regulated by PTEN over-expression was extracellular factors, which function as growth factor-binding or fibronectin-binding molecules related to cell adhesion or organ system development. In contrast, nuclear PTEN promoted the enrichment of genes in collagen-binding and integrin-binding, which may be related to blood vessel development or cell motility. KEGG pathway enrichment also confirmed that PTEN over-expression upregulated focal adhesion pathway, an important pathway that frequently disrupted in PTEN Hamartoma Tumor Syndrome (PHTS). Moreover, nuclear PTEN differentially regulated genes showed higher enrichment in immune response signaling pathways.

This unique gene expression profiling driven by NLS-PTEN indicated that besides the phosphatase activities, nuclear PTEN may have additional functions than merely inhibiting the PI3K signaling pathway. The transcriptome data showed potential downstream targets of PTEN in different subcellular localizations, which may lead to unique biological functions. More functional studies will be carried out to further validate how intracellular PTEN regulates the expression of these genes, and what are the signaling pathways involved in the regulatory processes.

Funding sources - This work was supported by the Hong Kong PhD Fellowship (PF12-13876) to WY. AC was supported by a General Research Fund grant (#460713), a Collaborative Research Fund grant (C014-14G) from the Hong Kong University Grants Committee, a Lo Kwee-Seong Biomedical Research Seed Fund, and the Brain & Mind Institute.

Note: This abstract was not presented at the meeting.

Citation Format: Yubing Wang, Andrew M. Chan. Effects of PTEN localization and phosphatase activity on gene expression profiling of glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5527. doi:10.1158/1538-7445.AM2017-5527
Abstract 5528: The protein phosphatase 2A regulatory subunit PR70 is a gonosomal melanoma tumor suppressor gene
Authors: Kempen Léon C.L. Van , Redpath Margaret , Elchebly Mounib , Klein Kathleen Oros , Papadakis Andreas , Willmott James , Scolyer Richard , Edqvist Per-Henrik , Ponten Fredrik , Schadendorf Dirk , Rjk Anke van , Michiels Stefan , Dumay Anne , Helbling-Leclerc Anne , Dessen Philippe , Wouters Jasper , Stass Marguerite , Greenwood Celia , Ghanem G. Elias , Oord Joost van den , Feunteun Jean , Spatz Alan
Published: July 2017

Male gender is independently and significantly associated with poor prognosis in melanoma of all clinical stages. The biological underpinnings of this sex difference remain largely unknown, but we hypothesize that gene expression from gonosomes may play an important role. The current study demonstrates that loss of the inactivated X chromosome in melanomas arising in females is strongly associated with poor distant metastasis-free survival, suggesting a dosage benefit of two X-chromosomes. The gonosomal PPP2R3B gene is located on the pseudo-autosomal region (PAR) of the X-chromosome in females and on the Y-chromosome in males. We observed that despite its location on the PAR that predicts equal dosage across genders, PPP2R3B expression is lower in males than in females, and is independently correlated with poor clinical outcome. PPP2R3B codes for the PR70 protein, a regulatory substrate recognizing subunit of PP2A, that decreases melanoma growth by negatively interfering with DNA replication and cell cycle progression through its role in stabilizing CDC6/CDT1 interaction that delays the firing of origins of DNA replication. As such, PR70 behaves functionally as an X-linked tumor suppressor gene.

Citation Format: Léon C.L. Van Kempen, Margaret Redpath, Mounib Elchebly, Kathleen Oros Klein, Andreas Papadakis, James Willmott, Richard Scolyer, Per-Henrik Edqvist, Fredrik Ponten, Dirk Schadendorf, Anke van Rjk, Stefan Michiels, Anne Dumay, Anne Helbling-Leclerc, Philippe Dessen, Jasper Wouters, Marguerite Stass, Celia Greenwood, G. Elias Ghanem, Joost van den Oord, Jean Feunteun, Alan Spatz. The protein phosphatase 2A regulatory subunit PR70 is a gonosomal melanoma tumor suppressor gene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5528. doi:10.1158/1538-7445.AM2017-5528
Abstract 5529: CD177 suppresses breast cancer progression by regulating the canonical WNT β-Catenin pathway
Authors: Kluz Paige N. , Kolb Ryan , Xie Qing , Borcherding Nicholas , Wang Linna , Andrade James P. De , Spanheimer Phillip M. , Li Wei , Gibson-Corley Katherine N. , Tao Andy W. , Sugg Sonia , Weigel Ronald J. , Zhang Weizhou
Published: July 2017

To better treat breast cancer patients, it is imperative to be able to predict breast cancer relapse and metastasis. Here, we identified the cluster of differentiation 177 (CD177) expression is positively correlated with relapse-free and metastasis-free survival of breast cancer patients. CD177 protein is highly expressed in normal breast epithelial cells and is significantly reduced in invasive cancers. Using mouse models, we found that CD177 suppresses breast cancer, primarily by forming a complex with E-Cadherin and b-Catenin at adherens junctions. The physical interaction prevents the activation of b-Catenin-mediated transcription by the canonical WNT signaling as demonstrated by a TOP-flash dual luciferase assay. In conclusion, we have identified CD177 as a novel breast cancer suppressor and uncovered a new regulatory mechanism for the canonical WNT/b-Catenin-mediated oncogenic signaling transduction.

Citation Format: Paige N. Kluz, Ryan Kolb, Qing Xie, Nicholas Borcherding, Linna Wang, James P. De Andrade, Phillip M. Spanheimer, Wei Li, Katherine N. Gibson-Corley, Andy W. Tao, Sonia Sugg, Ronald J. Weigel, Weizhou Zhang. CD177 suppresses breast cancer progression by regulating the canonical WNT β-Catenin pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5529. doi:10.1158/1538-7445.AM2017-5529
Abstract 553: Microsatellite genotyping enables precision lung cancer risk classification
Authors: Varghese Robin T. , Velmurugan Karthik Raja , Fonville Natalie C. , Garner Harold R.
Published: July 2017

There remains a large discrepancy between the known genetic contributions to cancer and that which can be explained by genomic variants, both inherited and somatic. Recently, understudied repetitive DNA regions called microsatellites have been identified as genetic risk markers for a number of diseases including various cancers (breast, ovarian and brain). In this study we demonstrate an integrated process for identifying and validating microsatellite based risk markers for lung cancer using data from the cancer genome atlas (TCGA) and the 1000 genomes project. Comparing whole exome germline sequencing data from 488 TCGA lung cancer samples to germline exome data from 390 control samples from the 1000 genomes project, we identified 119 potentially informative microsatellite loci. These loci (risk markers) were found to be able to distinguish between cancer and control samples with sensitivity and specificity ratios over 0.8. Then these loci, supplemented with additional loci from other cancers and controls, were evaluated using a custom target enrichment kit and sample-multiplexed nextgen sequencing. Thirteen of the 119 risk markers were validated using high-depth (579x±315) nextgen sequencing of 30 lung cancer and 89 control samples, resulting in sensitivity and specificity ratios were 0.90 and 0.94, respectively. When 8 loci harvested from the bioinformatic analysis of other cancers are added to the classifier, then the sensitivity and specificity rise to 0.93 and 0.97, respectively. Analysis of the genes harboring these loci revealed two genes (ARID1B and REL) and two significantly enriched pathways (chromatin organization and cellular response to stress) which suggest that the process of lung carcinogenesis is linked to chromatin remodeling, inflammation, and tumor microenvironment restructuring. We illustrate that high-depth nextgen sequencing enables a high precision microsatellite-based risk classifier.

Note: This abstract was not presented at the meeting.

Citation Format: Robin T. Varghese, Karthik Raja Velmurugan, Natalie C. Fonville, Harold R. Garner. Microsatellite genotyping enables precision lung cancer risk classification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 553. doi:10.1158/1538-7445.AM2017-553
Abstract 5534: Runx1 possesses anti-tumor activity and inhibits stemness in breast cancer cells
Authors: Hong Deli , J Andrew , Finstad Fritz, Kristiaan , Fitzgerald Mark P. , Stein Janet L. , Lian Jane , Stein Gary S.
Published: July 2017

Background: Despite advances in detection and therapies, breast cancer is still the most common malignant disease in women worldwide. Cancer stem cells (CSC) play an important role in formation, growth, drug-resistance and recurrence of tumors. Transcription factor Runx1 has received recent attention for its potential tumor suppressor activity in solid tumors. We previously showed that Runx1 represses the epithelial to mesenchymal transition in breast cancer cells. The objective of the present study is to investigate Runx1 functional effects on cellular activities, and its contribution to inhibiting CSC-like properties in breast cancer cells.

Methods: We generated Runx1 overexpression and Runx1 knockdown stable cell lines in both malignant MCF10AT1 and metastatic MCF10CA1a cells. Cells were analyzed for tumorsphere formation, protein (Western) and RNA (RT-qPCR) expression analysis. Cellular activity assays (cell proliferation, migration, and invasion) were performed to measure the tumorigenic potential of the cells. Xenograft studies were used to determine whether Runx1 overexpression in breast cancer cells represses tumor growth in vivo.

Results: Upon overexpressing Runx1, both MCF10AT1 and MCF10CA1a cells reduce their proliferation, migration and invasion, which support the concept that Runx1 acts as a tumor suppressor in breast cancer cells. Notably, overexpressing Runx1 inhibited the formation of tumorspheres and effectively reduced the stem-like population in these two breast cancer cells. These observations are consistent with the results of Runx1 knock-down in early stage ER positive MCF7 breast cancer cells which showed increased tumorsphere formation efficiency. Furthermore, Runx1 tumor suppressor activity was confirmed in mouse xenograft by showing that Runx1 overexpression inhibits tumor growth.

Conclusion: Our studies show that Runx1 inhibits proliferation, migration, and invasion, reflecting its tumor suppressor activity. Runx1 mediated reduction of tumorsphere formation suggests that Runx1 also contributes significantly to inhibit the population of the breast cancer stem cells. Therefore these studies offer Runx1 as a novel bio-therapeutic molecule for breast cancer intervention.

Citation Format: Deli Hong, Andrew J, Fritz, Kristiaan Finstad, Mark P. Fitzgerald, Janet L. Stein, Jane Lian, Gary S. Stein. Runx1 possesses anti-tumor activity and inhibits stemness in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5534. doi:10.1158/1538-7445.AM2017-5534
Abstract 5539: The phenotypic characterization of Lrig null mouse embryonic fibroblasts
Authors: Herdenberg Carl , Hedman Håkan
Published: July 2017

The LRIG gene family consists of LRIG1, LRIG2 and LRIG3. The most studied of these is LRIG1, which is a tumor suppressor gene. The LRIG1 protein is downregulated in various types of cancer and it negatively regulates receptor tyrosine kinases including the EGF- and PDGF-receptors. Lrig1 also regulates stem cell homeostasis in mouse skin and intestine. The functions of LRIG2 and LRIG3 are less known, but some findings imply that they might antagonize LRIG1. Intriguingly, the sole LRIG homolog in C. elegans, Sma-10, positively regulates BMP signaling. Thus, it is not known to what degree the LRIG proteins have the same, overlapping, or opposing functions, and the molecular mechanisms involved are poorly characterized. To elucidate the functions of the three LRIG proteins we generated a series of wildtype and LRIG-null (Lrig1-/-;Lrig2-/-;Lrig3-/-) mouse embryonic fibroblast (MEF) lines. These cell lines were created from homozygous Lrig1flox/flox;Lrig2flox/flox;Lrig3flox/flox mouse embryos. The FLOXed Lrig genes were then deleted with CRE-recombinase, thereby creating Lrig-null MEFs. ddPCR analysis showed that the Lrig-null MEF lines were contaminated with less than 1% of cells carrying wildtype Lrig3 alleles. Lrig-null MEFs showed a higher proliferation rate and migrated more in trans-well chambers than the wildtype cells. These cell lines will also be useful to investigate the importance of Lrig proteins for other cellular functions such as, signal transduction, lipidomics, transcriptomics, secretomics, metabolomics, and oxidative stress sensitivity. We also transduced the Lrig-null cells with individual inducible Lrig-genes thus creating a powerful system for the examination of the biology of the specific Lrig proteins.

Citation Format: Carl Herdenberg, Håkan Hedman. The phenotypic characterization of Lrig null mouse embryonic fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5539. doi:10.1158/1538-7445.AM2017-5539
Abstract 5543: Network guided modeling allows tumor type independent prediction of sensitivity to retinoic acid
Authors: Garattini Enrico , Bolis Marco , Fratelli Maddalena , Terao Mineko
Published: July 2017

Background: All-trans retinoic acid (ATRA) is a differentiating agent used in the treatment of acute-promyelocytic-leukemia and it is under-exploited in other malignancies despite its low systemic toxicity. A rational/personalized use of ATRA requires development of predictive tools allowing identification of sensitive cancer types and responsive individuals.

Materials and Methods: RNA-Sequencing data for 10080 patients and 33 different tumor-types were derived from the TCGA and Leucegene datasets and completely re-processed. The study was performed using machine learning methods and network analysis.

Results: We profiled a large panel of breast-cancer cell-lines for in vitro sensitivity to ATRA and exploited the associated basal gene-expression data to initially generate a model predicting ATRA-sensitivity in this disease. Starting from these results and using a network-guided approach, we developed a generalized model (ATRA-21) whose validity extends to tumor-types other than breast cancer. ATRA-21 predictions correlate with experimentally determined sensitivity in a large panel of cell-lines representative of numerous tumor-types. In patients, ATRA-21 correctly identifies APL as the most sensitive acute-myelogenous-leukemia subtype and indicates that uveal-melanoma and low-grade glioma are top-ranking diseases as for average predicted responsiveness to ATRA. There is a consistent number of tumor-types for which higher ATRA-21 predictions are associated with better outcomes.

Conclusions: In summary, we generated a tumor-type independent ATRA-sensitivity predictor which consists of a restricted number of genes and has the potential to be applied in the clinics. Identification of the tumor-types which are likely to be generally sensitive to the action of ATRA paves the way to the design of clinical studies in the context of these diseases. In addition, ATRA-21 may represent an important diagnostic tool for the selection of individual patients who may benefit from ATRA-based therapeutic strategies also in tumors characterized by lower average sensitivity.

Citation Format: Enrico Garattini, Marco Bolis, Maddalena Fratelli, Mineko Terao. Network guided modeling allows tumor type independent prediction of sensitivity to retinoic acid [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5543. doi:10.1158/1538-7445.AM2017-5543
Abstract 5544: New technologies to probe the systems glycobiology of cancer
Authors: Rudd Pauline M. , Hilliard Mark , Muniyappa Mohankumar , O'Flaherty Roisin , Saldova Radka
Published: July 2017

A robotic 384 well based platform was developed to release and label the human serum N-glycome. Improved separation of the glycan pool, based on hydrophilic interaction UPLC chromatography combined with mass spectrometry (Waters Corporation) and computer assisted data interpretation (NIBRT Glycobase) enabled us to build a database after assigning the detailed structures of more than 170 N-glycans.

The aim of the project was to use these technologies and databases to link glycosylation changes in individual patients’ serum with features of cancer and with changes in a range of other –omics data acquired from the same patients.

Alterations in glycosylation in various breast cancers were mapped to changes in the serum glycomes and aligned with genetic, transcriptomic and proteomic data. Pathway analysis showed strong associations between these glycan changes and the –omics data. This revealed that many of the glycan changes are directly associated with pathways involved in cancer metastasis.

Our next aim was to demonstrate the feasibility of collecting personalised data from individual patients from each of the –omics analyses to build up a dataset of the changing glycosylation over time. This opens up the possibility of linking these data to explore pathways of disease and, in particular, nodal points where the patient can no longer compensate for the effects of an altered pathway that is leading to disease.

To this end, single glycoproteins from 8 controls and 26 ovarian cancer patients were sequentially purified from 5ul of serum on affinity plates and the released glycans were analysed. Rather than the conventional way of looking at markers which compare a patients’ data with averages, the control can now be the blood of the patient themselves taken at an earlier time point.

The automation, sensitivity, quantitation and resolution of the new technology platforms coupled with dedicated software open up new possibilities for precision medicine and early intervention based on the biochemical profile of the patient.

References:

Association of N-glycosylation with breast carcinoma and systemic features using high-resolution quantitative UPLC. Saldova R, Asadi Shehni A, Haakensen VD, Steinfeld I, Hilliard M, Kifer I, Helland A, Yakhini Z, Børresen-Dale AL, Rudd PM. J Proteome Res. 2014 May 2;13(5):2314-27.

Serum N-glycan analysis in breast cancer patients–Relation to tumour biology and clinical outcome.

Haakensen VD, Steinfeld I, Saldova R, Shehni AA, Kifer I, Naume B, Rudd PM, Børresen-Dale AL,Yakhini Z. Mol Oncol. 2016 Jan;10(1):59-72.

 

Citation Format: Pauline M. Rudd, Mark Hilliard, Mohankumar Muniyappa, Roisin O'Flaherty, Radka Saldova. New technologies to probe the systems glycobiology of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5544. doi:10.1158/1538-7445.AM2017-5544