bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4980: Regulating the regulator: Long noncoding RNAs in the p53 network in colorectal cancer
Authors: Chaudhary Ritu , Li Xiao Ling , Lal Ashish
Published: August 2018

Thousands of long noncoding RNA (lncRNA) genes are transcribed from the human genome, yet the biology of the vast majority remains to be investigated. To identify lncRNAs that could be important, we functionally integrate lncRNAs into tumor suppressor pathways, more specifically the p53 pathway. We identified and functionally characterized two intergenic nuclear-retained lncRNAs that we named PINCR (p53 induced noncoding RNA) and PURPL (p53-upregulated regulator of p53 levels). Our results show that both PINCR (Chaudhary et al, eLife, 2017) and PURPL (Li et al, Cell Reports, 2017), function as pro-survival p53-regulated genes during DNA damage and in mouse xenografts. Mechanistically, PINCR and PURPL mediate the effects of p53 in colorectal cancer (CRC) cells by regulating p53 itself or by modulating the expression of a subset of p53 targets via interactions with unique proteins in the nucleus. Targeted deletion of PINCR in CRC cells significantly impaired G1 arrest and induced apoptosis to chemotherapeutic drugs. PINCR regulates the induction of a subset of p53 targets including BTG2, RRM2B and GPX1. Silencing these PINCR targets phenocopies the effect of loss of PINCR in response to DNA damage suggesting that they could be important downstream effectors of this lncRNA. Using CRISPR-Cas9-mediated knock-in of a single S1-tag at the 3'end of PINCR followed by an RNA pulldown approach that utilized endogenous S1-tagged PINCR, we found that PINCR associates with the enhancer region of these genes by binding to RNA-and DNA-binding protein Matrin 3 that, in turn, associates with p53. We show that PINCR is important for the recruitment of Matrin 3 to the enhancer region of these genes and for their complete p53-dependent induction after DNA damage. In addition, our unpublished data provide further insights on the molecular function of these lncRNAs in CRC. Collectively, our studies reveal critical roles of PINCR and PURPL in the p53 network and uncover unique modes by which lncRNAs can function.

Citation Format: Ritu Chaudhary, Xiao Ling Li, Ashish Lal. Regulating the regulator: Long noncoding RNAs in the p53 network in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4980.
Abstract 500: A p53/miR-30a/ZEB2 axis controls basal-like/triple-negative breast cancer aggressiveness
Authors: Damiano Valentina , Gennaro Alessandra di , Brisotto Giulia , Armellin Michela , Perin Tiziana , Zucchetto Antonella , Guardascione Michela , Spaink Herman P. , Doglioni Claudio , Snaar-Jagalska Ewa , Santarosa Manuela , Maestro Roberta
Published: August 2018

Inactivation of p53 plays a major role in the poor prognosis of basal-like /triple-negative breast cancers (TNBC). However, how p53 inactivation impinges upon TNBC aggressive phenotype is only partially defined. Here we report the identification of a novel axis involving p53, miR30a and ZEB2. We provide evidence that p53 affects the expression of ZEB2, a transcription factor involved in epithelial-mesenchymal transition (EMT), by relying on miR-30a. In particular, we found that p53 regulates miR-30a expression by direct promoter binding and that miR30a targets ZEB2. Finally, we provide evidence that the new p53/miR-30a/ZEB2 axis controls tumor cell invasion and migration and that reduced miR-30a expression correlates with p53 inactivation and dismal prognosis in human breast cancers. Overall, this study highlights the existence of a novel axis linking p53 to EMT via miR-30a, and adds a novel element to the complex network whereby p53 inactivation impinge upon tumor aggressiveness.

Citation Format: Valentina Damiano, Alessandra di Gennaro, Giulia Brisotto, Michela Armellin, Tiziana Perin, Antonella Zucchetto, Michela Guardascione, Herman P. Spaink, Claudio Doglioni, Ewa Snaar-Jagalska, Manuela Santarosa, Roberta Maestro. A p53/miR-30a/ZEB2 axis controls basal-like/triple-negative breast cancer aggressiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 500.
Abstract 5006: Kit-dependent tissue resident macrophage progenitors drive cancer progression
Authors: Pathria Paulina , Takahashi Hideyuki , Kaneda Megan , Varner Judith A.
Published: August 2018

Macrophages play a key role in promoting tumor growth and resistance to therapy. Here we show that tissue-resident as well as bone marrow-derived macrophages play critical roles in promoting tumor growth. Tissue-resident macrophages were recently shown to originate in the yolk sac or fetal liver during embryogenesis; these cells self-maintain in post-natal tissues independent of hematopoietic stem cells. Tissue-resident macrophages and bone marrow-derived macrophages rapidly accumulate in tumors where they play independent roles in promoting tumor growth. Tissue-resident macrophages are CD11b+Gr1-F4/80hiCX3CR1hiCCR2-Ki67+ cells that accumulate in tumors independently of trafficking receptors. In contrast, bone marrow-derived CD11b+Gr1+F4/80loCX3CR1loCCR2+ macrophages accumulate in tumors in an integrin α4β1/αLβ2 and CCR2 or CXCR4-dependent manner. Gene expression studies show that tumor-associated tissue-resident macrophages are highly proliferative, immune-suppressive myeloid cells that are less proangiogenic than bone marrow-derived macrophages. Our studies show that tumor cells induce the expansion of tissue-resident macrophage progenitor cells by secreting stem cell factor and mCSF. Here we identify a Kit/KitL-dependent tissue-resident macrophage progenitor that is abundant in tumors but not in normal tissues. Notably, tumor growth and colony-forming activity are significantly inhibited in mice treated with SCF and Kit inhibitors. Tumors adoptively transferred with tissue-resident macrophage progenitor cells exhibited a significant growth advantage over control tumors. Furthermore, Kit inhibitors synergize with other immune therapy regimens to suppress tumor growth. Our studies show that tissue-resident macrophage progenitors promote aggressive tumor growth that can be targeted by Kit/SCF inhibition.

Citation Format: Paulina Pathria, Hideyuki Takahashi, Megan Kaneda, Judith A. Varner. Kit-dependent tissue resident macrophage progenitors drive cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5006.
Abstract 5016: A unique ex vivo drug evaluation model: 3D co-cultured system with tumor, stroma and blood microvessels
Authors: Takahashi Yuki , Tsukamoto Kei , Kitano Shiro , Irie Shinji , Matsusaki Michiya , Nagayama Satoshi , Katayama Ryohei , Shinozaki Eiji , Fujita Naoya
Published: August 2018

Background; Two dimensional single-layer culture still remains the preferred platform for most laboratory preclinical studies, although interaction between cancer and stromal cells has been well reported to be important in tumor progression and resistance against therapies. In order to mimic the patient tumor tissues, ex vivo model which recaptures the tumor microenvironment is required.

Methods; Layered 3D stromal tissues were produced by culturing normal human dermal fibroblasts (NHDFs) and human umbilical vein endothelial cells (HUVECs) coated with extra-cellular matrix (ECM) and natural polysaccharide, namely collagen and heparin. The layered 3D stromal tissues and overlaid tumors were morphologically characterized by HE stain, immunohistochemistry (IHC) and immunofluorescence (IF). Furthermore, drug sensitivity assays were conducted using popular colorectal cancer cell lines, and patient-derived cell lines (PDCs) established in the laboratory of Japanese Foundation for Cancer Research. Cancer cell viability was evaluated by fluorescent labeling, enzymatic dissociation and cell counting analysis. IF with cancer specific markers and imaging analyses were also performed.

Results; The 3D stromal tissues including CD31 positive luminal structure were multi-layered (approximately 20 layers), and formation of microvascular network was observed within several days. In comparison with 2D mono-culture or 3D mono/co-cultured spheroid model, decreased drug sensitivities were represented in our 3D co-cultured model. In the simultaneous treatment with cytotoxic anticancer agents and molecular targeted drugs, dose-responses were significantly different between the2D and 3D models.

Conclusion; We developed the layered 3D stromal tissue culture system including blood micro-vessels. Drug evaluation with the co-cultured tumors may reflect the drug sensitivity of cancer cells in vivo. Our unique 3D ex vivo model represents a valuable tool for drug development in a fully human cell and matrix microenvironment, and thus testing patient-derived cells and approved compounds also enable better prediction their efficacy.

Citation Format: Yuki Takahashi, Kei Tsukamoto, Shiro Kitano, Shinji Irie, Michiya Matsusaki, Satoshi Nagayama, Ryohei Katayama, Eiji Shinozaki, Naoya Fujita. A unique ex vivo drug evaluation model: 3D co-cultured system with tumor, stroma and blood microvessels [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5016.
Abstract 5035: Identification of novel therapeutics of bladder cancer cells through upregulation of SPARC expression in using 2D cell cultures and 3D bladder organoids
Authors: Patel Chirayu M. , Ivan Cristina , Atala Anthony , Said Neveen
Published: August 2018

We have recently reported the tumor suppressor effect of SPARC in urinary bladder cancer. We have shown that the loss of SPARC in bladder cancer resulted accelerated bladder carcinogenesis and metastasis multiple pathways involving cell cycle deregulation, inflammation and angiogenesis. We have also reported that SPARC protein expression significantly decreased in the cancerous compartment in advanced human bladder cancer as well as in carcinogen-induced murine urothelial cancer. However, the mechanism of the downregulation of SPARC expression in cancer in general and in bladder cancer in particular is still unraveled. The purpose of our study is to identify therapeutic candidates as regulators of SPARC expression in bladder cancer. We developed an in vitro 3-D bladder model system “bladder organoids” that comprises the normal and cancerous urothelial cell, bladder smooth muscle cells, fibroblasts and endothelial cells. We differentially expressed fluorescent SPARC promoter reports in the cancerous and non-cancerous cells to screen for the differential expression of SPARC in each cell type using FDA-approved and natural product drug libraries and to determine the cytotoxicity on cancerous vs non-cancerous cells. Real time monitoring of changes in SPARC expression was done by measuring fluorescence intensity using IncuCyte® ZOOM and InCell Analyzer 1000 Live Imaging Analysis Systems. We identified drugs that consistently exhibited dose-dependent augmentation or down-regulation of SPARC expression in cancer cells. Our screens using the identified novel up-regulators of SPARC expression that differentially reduce the viability of cancer cells and spare non-cancer cells in the organoids. Our efforts produced a platform that mimics bladder cancer microenvironment that can be adapted for throughput screening for targeted personalized therapies.

Citation Format: Chirayu M. Patel, Cristina Ivan, Anthony Atala, Neveen Said. Identification of novel therapeutics of bladder cancer cells through upregulation of SPARC expression in using 2D cell cultures and 3D bladder organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5035.
Abstract 5036: Visualization of cancer-derived exosome-mediated angiogenesis in a layered 3D stromal tissue model
Authors: Kitano Shiro , Tsukamoto Kei , Morimura Rii , Irie Shinji , Shinozaki Eiji , Yamaguchi Kensei
Published: August 2018

Background: Exosomes are small vesicles of sizes ranging from 30 to 150 nm that originate from the endosomal luminal membrane and are constantly released by fusion with cell membranes. Several studies have revealed that exosomes play an important role in mediating local and systemic cellular communication through the transmission of nucleic acid materials and information in the form of proteins. Here, we demonstrated a unique layered 3D stromal tissue model of visualization of promoting angiogenesis by exosomes in colorectal cancer (CRC) patients.

Method: The CRC specimens of exosome were obtained from 7 patients in stage IV. Isolation of total exosome was performed according to standard procedure by ultracentrifuge. Layered 3D stromal tissue model was constructed with human umbilical vein endothelial cells (HUVEC) and normal human dermal fibroblasts. Cells were aggregated by heparin and collagen to suspension under cationic condition. The cell suspension was seeded to transwell inserts membrane at a density of 2×106. Media were changed after 24h, and serum, exosome or supernatant after ultracentrifuge of serum (SAUS) was added to the 3D stromal tissue after 6 days. The isolated exosomes were diluted with an equal volume of PBS buffer to the serum sample. After cultivation, the tube formation of HUVEC was stained with Immunofluorescence, and the degree of hypervascularity was compared with healthy volunteers.

Result: Except for one serum specimen, hypervascularization was observed in the 3D stromal tissue model in an additive-dependent manner. Especially when exosome was added, hypervascularization was observed in the layered 3D stromal tissue model more efficiently than serum in all specimens (see Table). Interestingly, the frequency of hypervascularization was drastically reduced in the SAUS.

Conclusion: Our data encourage that this layered 3D stromal tissue model may be applicable to cancer diagnosis using exosomes from CRC patients.

Table. Frequency of hypervascularization by exosomes derived from CRC5μl10μl50μlExosome5/7 (71.4%)7/7 (100.0%)7/7 (100.0%)Serum4/7 (57.1%)6/7 (85.7%)6/7 (85.7%)SAUS1/7 (14.3%)0/7 (0.0%)0/7 (0.0%)

Citation Format: Shiro Kitano, Kei Tsukamoto, Rii Morimura, Shinji Irie, Eiji Shinozaki, Kensei Yamaguchi. Visualization of cancer-derived exosome-mediated angiogenesis in a layered 3D stromal tissue model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5036.
Abstract 5053: Mlh1 deficiency increases the risk of hematopoietic malignancies post low- and high-LET radiation exposure
Authors: Patel Rutulkumar , Gerson Stanton L. , Welford Scott M.
Published: August 2018

Introduction/Aim: Natural sources of radiation in space include galactic cosmic rays (GCR), solar energetic particles and trapped energetic particles in a planetary magnetic field. These sources are difficult to shield, thus posing significant health risks to astronauts on long-term inter-planetary missions. During space travel, genomic instability is a major concern where astronauts are exposed to potent sources of ionizing radiation, namely GCR consisting of high energy and charged atomic nuclei. In particular, hematopoietic stem cells (HSCs) are susceptible to internal and external stresses that threaten the integrity of the cell, and accumulation of damage can lead to HSCs dysfunction and oncogenesis. Recent data has demonstrated that humans accumulate microsatellite instability with acquired loss of MLH1 protein in HSCs as a function of age. Therefore, we hypothesized that high-LET (linear energy transfer) radiation characteristic of the GCR that will confront astronauts on space missions will damage HSCs and contribute to induction and progression of hematopoietic malignancies. Methods: To study this hypothesis, we employed a DNA mismatch repair deficient mouse model (Mlh1+/-) to study the effects of low-LET γ-ray vs high-LET 56Fe ion radiation on HSCs of potential astronaut population. In vitro colony forming unit assays and in vivo complete blood count (CBC) plus competitive repopulation assays were carried out to understand harmful impact of radiation and Mlh1 deficiency on HSCs functions. In addition, mice were followed up to 18 months post irradiation to observe HSC malignancies in Mlh1+/+ and Mlh1+/- mice. Results: HSC short- and long-term functional assays showed defects in HSCs/HPCs function caused by irradiation, but not depending on Mlh1 status. CBC 5 and 9 months post irradiation demonstrated no impact of irradiation or Mlh1 status on HSC differentiation. However, 56Fe-ion irradiated Mlh1+/- mice showed a significant higher incidence of lymphomagenesis compared to γ-rays irradiated and sham-irradiated Mlh1+/- mice. In addition, immunohistochemistry analysis of lymphomas displayed significant higher incidence of T-cell rich B-cell lymphomas. Conclusion: Thus, the data show that MMR defects in HSCs leads to sensitization to radiation induced hematopoietic malignancy, and that radiation quality effects exacerbate the sensitivity. The findings could have profound effects on astronaut screening and designing better mitigators for space missions.

Citation Format: Rutulkumar Patel, Stanton L. Gerson, Scott M. Welford. Mlh1 deficiency increases the risk of hematopoietic malignancies post low- and high-LET radiation exposure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5053.
Abstract 5067: b1-Integrin promotes inflammatory cytokine-induced vascular leakage
Authors: Kiss Elina A. , Hakanpää Laura , Jacquemet Guillaume , Miinalainen Ilkka , Lerche Martina , Guzman Camilo , Mervaala Eero , Eklund Lauri , Ivaska Johanna , Saharinen Pipsa
Published: August 2018

Increased capillary leakage is a potentially life-threatening condition associated with sepsis and cytokine release syndrome (CRS) that develops as a side effect of novel chimeric antigen receptor (CAR) T-cell cancer immunotherapies. CRS and sepsis are characterized by immune activation, resulting in elevated circulating levels of inflammatory cytokines including IL-6 and IL-1β, and thrombin that promote vascular leakage and the development of hypovolemic shock and multiorgan failure. However, molecular mechanisms behind capillary leakage remain unknown, and importantly, targeted approaches for the management of capillary leakage are limited. Here, we found that β1-integrin acts as a critical universal mediator of endothelial permeability induced by various inflammatory agents, including LPS, IL-6, IL-1β and thrombin. In a mouse model of LPS-induced systemic inflammation, resulting in increased inflammatory cytokine production and vascular leakage, function blocking antibodies against β1-integrin prevented vascular leakage and improved integrity of vascular endothelium, leading to protection from LPS-induced cardiac failure. β1-integrin blocking antibodies were effective in reducing vascular leakage even when administered after the onset of systemic inflammation and increase in the serum levels of IL-6, IL-1β and TNF-α. By using an in vitro model, we found that inflammatory agents generated mechanical stress, in a β1-integrin dependent manner, leading to endothelial cell contractility and permeability. Conclusively, our data indicates that β1-integrin promotes vascular leakage in inflammation, and that targeting this mechanism may have therapeutic utility for vascular stabilization in CRS and capillary leak syndromes associated with immune activation and systemic inflammation.

Citation Format: Elina A. Kiss, Laura Hakanpää, Guillaume Jacquemet, Ilkka Miinalainen, Martina Lerche, Camilo Guzman, Eero Mervaala, Lauri Eklund, Johanna Ivaska, Pipsa Saharinen. b1-Integrin promotes inflammatory cytokine-induced vascular leakage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5067.
Abstract 5071: The functional relevance of Atypical Chemokine Receptor 1 (ACKR1/DARC) genetic isoforms in breast cancer
Authors: Jenkins Brittany D. , Martini Rachel N. , Brown Inasia , Davis Melissa B.
Published: August 2018

Chemokines and their receptors can interact to affect the host's anti-tumor response by influencing the migration of host immune cells indirectly via a chemokine gradient. The regulation and function of these receptors is paramount in ensuring an appropriate immune response to an inflammatory stimulus, especially in the case of cancer. One such receptor, Atypical Chemokine Receptor 1 (ACKR1/DARC), is a genetically diverse transmembrane GPCR that can buffer pro-inflammatory CXC- and CC- chemokine activity in circulation and within tissues, influencing leukocyte migration. While the basic immunological function of this receptor is known, the specific function of its two genetic isoforms, A and B, is not. These isoforms are produced by two alternative promoters and distinct mRNA splicing events. Isoform A is not split by introns, even in the untranslated regions, and offers homology to CXCL8. The alternative splice variant, Isoform B, does contain an intron, and follows the GT-AG splicing rule, including a novel exon coding for an additional 7 amino acids. This localizes to the N-terminus, making up part of the extracellular binding region of the receptor. We aim to determine if the two major ACKR1 isoforms have distinct functional abilities to bind or sequester pro-inflammatory chemokines in epithelial breast cell lines. By creating isoform specific overexpression and knockout constructs, and transiently transfecting the DNA into our subtype specific cell lines, we can use an ELISA assay to determine chemokine concentration in the media, and measure differences based on isoform specificity. Using immunofluorescence, we first show that ACKR1 and our two proinflammatory chemokines, CCL2 and CXCL8, are expressed on the surface of epithelial cells and colocalize. We then indicate that there is an isoform specific difference in chemokine concentration between different breast cancer subtypes, in addition to differences seen in overexpression and knockout cell lines. Determining the functionality of these two isoforms can help us better understand ACKR1's role in the immune system, and its potential role as a prognostic marker in breast cancer.

Citation Format: Brittany D. Jenkins, Rachel N. Martini, Inasia Brown, Melissa B. Davis. The functional relevance of Atypical Chemokine Receptor 1 (ACKR1/DARC) genetic isoforms in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5071.
Abstract 511: HPV mediates HNSCC carcinogenesis by exosomal exclusion of tumor suppressive miRNAs
Authors: Khullar Madhu , Singh Varinder , Dogra Nilambra , Sannigrahi Malay , Panda Naresh
Published: August 2018

Background: Exosomes are small vesicles between 40-150 nm in diameter that are secreted by various cell types, including tumor cells. Exosomes of cancer cells are known to be involved in tumor pathology and have altered expression of microRNAs (miRNAs). However, the effect of HPV-16 (Human Papilloma Virus-16) infection on miRNA content of exosomes released by infected HNSCC cells has not yet been explored.

Materials & Methods: Exosomes were isolated from media of cultured HPV positive 93VU-147T and HPV negative UPCI:SCC 116 HNSCC cells and were characterized by Transmission Electron Microscopy. Presence of exosomal marker CD63 was confirmed using western blotting. Small RNA seq of exoRNA of HPV positive and HPV negative cells was carried out. For validation of identified miRNAs, HPV16 E6E7 was cloned into pcDNA3.1(-ve) vector and was transfected into SCC116 cells and stable cells were selected using G418. Differentially expressed miRNAs were validated in cells and exosomes using Real Time PCR.

Results: Seven miRNAs 6724-5p, 6087, 4497, 4792, 4508, 199a-3p and 199b-3p were found to be significantly upregulated in HPV positive HNSCC exosomes as compared to HPV negative HNSCC exosomes from NGS data. Upregulation of these miRNAs was further confirmed in exosomes derived from HPV positive 93VU-147T cells and SCC116 E6E7 cells as compared to HPV negative cells (SCC 116 (3.1-ve) (vector only transfected SCC 116) cells. Expression of these miRNAs was significantly downregulated in 93VU14T and SCC116 E6E7 (HPV +ve) cells as compared to HPV negative cells. Three of these miRNAS (4792, 199a-3p and 199b-3p) are known tumor suppressor miRNAs. miRNA 6724-5p, a novel miRNA was found to be down regulated the most in HPV +ve cells. We identified CBX6, PDK2 and FOXK1 which are known to be involved in carcinogenesis, as its target genes through Targetscan. Expression of these genes was found to be significantly upregulated in HPV positive 93VU-147T cells and SCC116 E6E7 cells as compared to HPV negative cells indicating tumor suppressive role of miRNA 6724-5p in carcinogenesis.

Conclusion: Our results suggest that HPV may mediate HNSCC carcinogenesis by exosomal exclusion of tumor suppressive miRNAs.

Citation Format: Madhu Khullar, Varinder Singh, Nilambra Dogra, Malay Sannigrahi, Naresh Panda. HPV mediates HNSCC carcinogenesis by exosomal exclusion of tumor suppressive miRNAs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 511.
Abstract 5127: Eradication of the gut microbiota reduces cancer burden in multiple models by modulating the immune system
Authors: Sethi Vrishketan , Kurtom Saba , Tarique Mohd , Giri Bhuwan , Garg Bharti , Lavania Shweta , Ferrantella Anthony , Malchiodi Zoe , Hellmund Leonor , Jacob Harrys Charles , Dawra Rajinder , Banerjee Sulagna , Roy Sabita , Ramakrishnan Sundaram , Saluja Ashok , Dudeja Vikas
Published: August 2018

Introduction: Bacteria residing in the human body outnumber ‘human' cells and most of these bacteria exist in the gut. This gut microbiome forms a unique and dynamic metagenome which changes with nutrition and even disease state. A growing body of evidence has implicated the role of the gut microbiome in modulating our immune system. We aimed to investigate this association in murine models of cancer. Methods: C57BL/6 mice were either orally given saline or a gut sterilizing cocktail of poorly absorbable broad-spectrum antibiotics. These mice were used to model multiple cancers, viz. subcutaneous injection of melanoma cells derived from Tyr::CreER; Braf V600E/+;Ptenlox5/lox5 mice, subcutaneous injection of pancreatic cancer cells derived from KPC (Kras LSL.G12D/+; p53 R172H/+ ;Pdx::Cre) mice, intrasplenic injection of KPC pancreatic cancer cells and intrasplenic injection of B16-F10 melanoma cells. The tumors were immunophenotyped through flow cytometry and immunostained for multiple antigens. Mice plasma was used for chemokine analysis. To confirm the role of immunity, subcutaneous experiments were repeated in mice carrying a Rag1tm1Mom mutation (RAG1 knockout), thereby having an immature adaptive immune system. Results: Gut microbiome depletion reduced tumor burden across all four subcutaneous and hepatic metastases models. This effect disappeared when experiments were repeated in immunosuppressed RAG1 knockout mice. Antibiotic-treated tumors had increased TNF-secreting macrophages and NKT cells as well a higher number of iNOS staining cells in the microenvironment. Plasma from antibiotic-treated tumor-bearing mice had decreased levels of CXCL12, a chemokine known to cause tumor immunosuppression. Conclusion: Eradication of the gut microbiota decreases tumor burden by activating the anti-cancer immune system

Citation Format: Vrishketan Sethi, Saba Kurtom, Mohd Tarique, Bhuwan Giri, Bharti Garg, Shweta Lavania, Anthony Ferrantella, Zoe Malchiodi, Leonor Hellmund, Harrys Charles Jacob, Rajinder Dawra, Sulagna Banerjee, Sabita Roy, Sundaram Ramakrishnan, Ashok Saluja, Vikas Dudeja. Eradication of the gut microbiota reduces cancer burden in multiple models by modulating the immune system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5127.
Abstract 514: Lncrna uca1 interacts directly with angiomotin to activate Hippo-YAP signaling in epithelial ovarian cancer
Authors: Lin Xianzhi , Spindler Tassja J. , Gayther Simon A. , Lawrenson Kate
Published: August 2018

The long noncoding RNA (lncRNA) urothelial cancer associated 1 (UCA1) is upregulated in multiple cancers, including epithelial ovarian cancer (EOC). Our present study aims to dissect the functional and mechanistic role of UCA1 in EOC. We first explored the effects of stable UCA1 knockout mediated by CRISPR/Cas9 genome editing. The effects of UCA1 loss were more profound in vivo, as UCA1 knockout significantly impaired tumor growth in a mouse xenograft model. Pathways downstream of UCA1 were catalogued using reverse phase protein arrays, which revealed that YAP signaling is activated in UCA1-overexpressing ovarian cancer cells. To characterize the UCA1 interactome, in vivo RNA antisense purification (UCA1-iRAP) experiments were performed using a modified ChIRP and RAP protocol to isolate in vivo RNA direct interactomes based on hybridization of short (20 base) biotinylated probes at a physiologically relevant temperature (37°C). UCA1-iRAP was performed using an EOC cell model, and proteins extracted were profiled by mass spectrometry (MS). Altogether, 19 unique proteins were reproducibly detected by UCA1-iRAP-MS. Interestingly 16 of these UCA1-associated proteins were not previously known RNA-binding proteins. Integration of UCA1-iRAP-MS data with multilayered functional characteristics of UCA1 knockout/knockdown identified angiomotin (AMOT), a known Hippo-YAP signaling modulator, as potential interacting partner for UCA1. The interaction between UCA1 and AMOT was confirmed using both Western blotting for AMOT using UCA1-iRAP proteins, and RT-qPCR for UCA1 RNA after AMOT immunoprecipitation in 3 independent EOC cell lines. Knockdown experiments showed that AMOT is required for UCA1 to activate the expression of YAP target genes, including CYR61 and AXL. Our results indicate that UCA1 interacts directly with AMOT to promote cell growth by activation of Hippo-YAP signaling in EOC, and for the first time identify UCA1 as a novel upstream regulator of YAP signaling. Moreover, the optimized iRAP method we developed in this study can be readily applied to characterizing the protein, DNA, and RNA interactomes of noncoding or coding transcripts implicated other complex traits.

Citation Format: Xianzhi Lin, Tassja J. Spindler, Simon A. Gayther, Kate Lawrenson. Lncrna uca1 interacts directly with angiomotin to activate Hippo-YAP signaling in epithelial ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 514.
Abstract 5140: Lipoxins and their role in Kaposi's sarcoma-associated herpesvirus (KSHV) infection and pathogenesis
Authors: Sharma-Walia Neelam , Chandrasekharan Jayashree A.
Published: August 2018

Lipoxins are host anti-inflammatory molecules, which play a vital role in restoring tissue homeostasis. The efficacy of lipoxins and their analog epilipoxins in treating inflammation and related diseases have been well documented. Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL) are two well-known inflammation-related diseases caused by Kaposi's sarcoma-associated herpesvirus (KSHV). Controlling inflammation is one of the strategies adopted to treat KS and PEL, a primary motivation for exploring and evaluating the therapeutic potential of using lipoxins. Our study demonstrates how KSHV manipulates and downregulates the secretion of the anti-inflammatory lipoxin A4 in host cells and the viral factors involved in this process using in vitro KS and PEL cells as models. Presence of lipoxin receptor [ALX/Formyl peptidyl receptor (FPR)] in human KS patient tissue sections and in vitro KS and PEL cell models offers a novel possibility for treating KS and PEL with lipoxins. Treating de novo KSHV-infected endothelial cells with lipoxin and epilipoxin creates an anti-inflammatory environment by decreasing levels of NFκB, AKT, ERK1/2, COX-2, and 5-lipoxygenase. Lipoxin treatment on CRISPR/CAS9 technology-mediated ALX/FPR gene-deleted U2OS cells revealed the importance of the lipoxin receptor ALX for effective lipoxin signaling. A viral miRNA cluster was identified as the primary factor contributing to the downregulation of lipoxin A4 secretion in host cells. The KSHV miRNA cluster probably targets enzyme 15-lipoxygenase, which is involved in lipoxin A4 synthesis. Lipoxin treatment in KSHV-infected cells has been shown to reduce cell proliferation and cell survival. Latency factor LANA-1 levels were found to decrease on lipoxin and epilipoxin treatment. Several pro-apoptotic gene levels were upregulated on lipoxin treatment. Several host transcription factors were found to play an important role in regulating KSHV life cycle. This study provides a new insight into the treatment of KS and PEL using nature's own anti-inflammatory molecule, lipoxin.

Citation Format: Neelam Sharma-Walia, Jayashree A. Chandrasekharan. Lipoxins and their role in Kaposi's sarcoma-associated herpesvirus (KSHV) infection and pathogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5140.
Abstract 5141: Streptococcus gallolyticus in colorectal cancer development
Authors: Xu Yi , Kumar Ritesh , Taylor John , Xu Juan
Published: August 2018

Streptococcus gallolyticus subsp. gallolyticus (Sg) has been reported to have a strong clinical correlation with colorectal cancer (CRC) for several decades. Our recent data indicates that Sg is able to actively promote the development of colon tumor and thus is also functionally important to CRC. Further investigations into the Sg-CRC connection revealed that there are variations among Sg strains with respect to their ability to stimulate target host cell proliferation and tumor growth. The ability of Sg to adhere to colon cancer cells and to colonize colonic epithelium in vivo appears to be important. The results suggest that direct interactions between specific Sg factors and colonic epithelial receptors/structures are required to trigger downstream signaling events leading to increased host cell proliferation. Furthermore, we observed that the effect of Sg is also dependent on the host genetic background, suggesting a dynamic and complex interactions between Sg and the host. In addition to the requirement for beta-catenin signaling as we previously reported, we observed that Sg activates yes-associated protein 1 (YAP1), a downstream effector in the Hippo signaling pathway. YAP1 activation is required for Sg to stimulate target host cell proliferation. YAP1 and the Hippo pathway are known to be involved in the development CRC. Bacterial activation of YAP1 has not been described before. Thus, our results suggest a novel mechanism by which bacteria contribute to the development of cancer. Studies are on-going to further investigate the molecular details of this mechanism.

Citation Format: Yi Xu, Ritesh Kumar, John Taylor, Juan Xu. Streptococcus gallolyticus in colorectal cancer development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5141.
Abstract 5162: Oligodendrocytes might up-regulate the invasiveness of glioblastoma cells via Angiopoietin-2 signaling pathway
Authors: Kawashima Toshiyuki
Published: August 2018

Background & Aims: Glioblastoma (GBM, WHO Grade IV) is considered as the most lethal neoplasm of all solid cancers due to its inherent intensive invasiveness. GBM may arise de novo, or following progression from lower grade gliomas. Recently, solid tumor progression has been recognized as the product of an evolving crosstalk between the cancer cells and its surrounding glial cells. The intensive invasion activity of GBM cells might be regulated by surrounding normal cells such as oligodendrocytes or fibroblasts. In this study, we evaluated the interactive factors between GBM cells and normal glial cells to determine the role of oligodendrocytes in regulating the invasiveness of glioblastoma cells. Methods: Two GBM cell lines, T98G and U251, were used. Two oligodendrocyte cell lines, ODC1 and ODC2, were derived one each from surgical tissues of patients with low-grade glioma (WHO Grade II). Two fibroblasts cell lines, GF1 and GF2, were derived one each from surgical tissues of patients with GBM. Oligodendrocytes and fibroblasts were confirmed by immuno-histochemical staining. The invasive property of GBM cells was analyzed in the presence or absence of conditioned medium from oligodendrocytes or fibroblasts by wound-healing assay and Boyden chamber invasion assay. The proliferation ability of GBM cells was examined by MTT assay. Subsequently Cytokine array was used to examine the cytokines and growth factors in the conditioned medium from oligodendrocytes or fibroblasts. Results: Oligodendrocyte cells, ODC1 and ODC2 cells, significantly (p<0.01) increased the migration and invasion ability of GBM cells, T98G and U251. In contrast, fibroblasts, GF1 and GF2 cells, did not affect the migration and invasion ability of GBM cells. Cytokine array indicated that Angiopoietin-2 was found in the conditioned medium from oligodendrocytes, but not that from fibroblasts. Angiopoietin-2 significantly (p<0.01) increased the invasiveness of GBM, but not their proliferation ability.

Conclusion: Oligodendrocytes might up-regulation of the invasiveness of GBM cells via angiopoietin-2 signaling pathway. Hence, angiopoietin-2 can be considered as a promising target for the treatment of malignant gliomas.

Citation Format: Toshiyuki Kawashima. Oligodendrocytes might up-regulate the invasiveness of glioblastoma cells via Angiopoietin-2 signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5162.
Abstract 5163: Rab11 regulates focal adhesion kinase dynamics in sarcoma cell migration
Authors: Kao Ling-Yi , Wei Wan-Chen , Chiu Hsiang-Ling , Wu Yi-Che , Chao Wei-Ting
Published: August 2018

Focal adhesion (FA) turnover has been demonstrated to play an important role in controlling cell migration and cancer metastasis. FA disassembly was shown to be regulated by endocytosis during cell migration. Rabs are a kind of GTPase that participate in various cellular vesicle transports; however, whether the Rabs play a role in mediating vesicle transport for FA formation is not clear. This study focuses on Rab11, which plays a role in endosome recycling, and examines whether Rab11 has regulatory function in FA formation during cell migration. Wild-type and deficient Rab11 were transfected into human HT1080 fibrosarcoma cells, the cell migration ability was determined by trans-well assay, the localization of Rab11 and focal adhesion molecules were monitored by confocal microscopy and the in vivo study was carried out with subcutaneous xenograft mouse model to evaluate the effect of Rab11 on tumor growth. Results showed Rab11 deficiency inhibited sarcoma cell migration; the Rab11 was also found colocalized with recycled β1 integrin and affected focal adhesion formation. The live image results demonstrated the inactive GDP form Rab11 has no effect on random cell migration; however, the Rab11 knockdown inhibited random cell migration. We further used immunofluorescence and immunoprecipitation and identified the physical interaction of Rab11 and FAK. In the in vivo study, wild-type Rab11 transfected cells increased the tumor volume in xenograft mouse, but not in Rab11-deficient cells. Taken together, the results suggested Rab11 affected cell migration by regulating focal adhesion dynamics through integrin recycling and the effect of Rab11 may be through protein-protein interaction other than GTP activation.

Citation Format: Ling-Yi Kao, Wan-Chen Wei, Hsiang-Ling Chiu, Yi-Che Wu, Wei-Ting Chao. Rab11 regulates focal adhesion kinase dynamics in sarcoma cell migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5163.
Abstract 5171: Tfap2b overexpression contributes to tumor growth & progression of thyroid cancer through AKT&VEGF/PEDF signaling pathway
Authors: Fu Xiaoyan , Yang Zhongyuan , Chen Shuwei
Published: August 2018

TFAP2 is a family of transcription factors implicated in many aspects of development.TFAP2B is a member of the AP2 transcription factor family, which orchestrates a variety of cell processes. However, the roles of TFAP2B in regulating thyroid carcinogenesis remain largely unknown. Here, we investigated the regulatory effects of TFAP2B on thyroid cancer growth & identified the underlying mechanisms of actions in thyroid cancer cells.We examined the expression of TFAP2B in thyroid cancer cell lines & tumor tissues. We also analyzed the TNM staging of TFAP2B in thyroid cancer. Then we investigated the molecular mechanisms by which TFAP2B knockdown or overexpression regulated thyroid cancer cell growth, angiogenesis & apoptosis, & further confirmed the role of TFAP2B in tumor growth in a thyroid cancerxenograft mouse model.TFAP2B was expressed in thyroid cancer lines &tumortissues,especially in anaplastic thyroid cancer. Strong TFAP2B expression showed a positive correlation with the poor differentiation of patients with thyroid cancer . TFAP2B knockdown by siRNA significantly inhibited cell growth & induced apoptosis in thyroid cancer cells in vitro & in a thyroid cancersubcutaneous xenograft model, whereas TFAP2B overexpression promoted cell growth. The observed regulation of cell growth was accompanied by the TFAP2B-mediated modulation of the VEGF/PEDF-dependent signaling pathways in thyroid cancer cells.These results indicate that TFAP2B plays a critical role in regulating thyroid cancer growth & could serve as a promising therapeutic target or diagonistic biomarker for thyroid cancer .

Citation Format: Xiaoyan Fu, Zhongyuan Yang, Shuwei Chen. Tfap2b overexpression contributes to tumor growth & progression of thyroid cancer through AKT&VEGF/PEDF signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5171.
Abstract 5182: Activity and clinical relevance of autotaxin and lysophosphatidic acid pathways in high-grade serous carcinoma
Authors: Onallah Hadil , Trope Claes G , Falkenthal Thea E. Hetland , Davidson Ben , Reich Reuven
Published: August 2018

Lysophosphatidic acid (LPA) is a bioactive phospholipid with mitogenic and growth factor-like activities affecting cell invasion, cancer progression and drug resistance. It is produced mainly by Autotaxin and acts on six G protein-coupled receptors, LPAR1-6. LPA has recently been implicated as a growth factor present in ascites of ovarian cancer patients. However, mitogenic pathways stimulated by LPA via its receptors are still far uncharacterized. Here we show that three LPA receptors are involved in the progression of the malignancy by the activation of both AKT and ERK pathways. We analyzed a set of 155 samples of high grade serous ovarian carcinoma (HGSC) from the three different anatomic sites of the disease. Results show that LPAR2 mRNA was overexpressed in HGSC cells in effusions compared to solid lesions, with opposite findings for LPAR3 and LPAR6 mRNA and ATX protein that was found mainly in exosomes. LPAR3 levels were significantly higher in pre-chemotherapy effusions compared to post-chemotherapy specimens (p=0.025). Higher expression of LPAR1 (p=0.037), LPAR2 (p=0.025) and LPAR5 (p=0.021) was significantly associated with shorter overall survival. Furthermore, we combined 3D (spheroids) and 2D cell culture models with gene editing using the CRISPR/Cas9 method for LPAR2, LPAR3 and LPAR6 and tested the effects of their silencing in vitro using OVCAR3 and ES2 cell lines. The p-ERK/ERK ratio was significantly elevated in LPAR3KO OVCAR3 cells cultured both in 2D and 3D form. Yet, LPAR2KO OVCAR3 cells expressed low levels of p-ERK/ERK in the 3D form, suggesting that LPAR3 inhibits p-ERK activity downstream, while LPAR2 activates it. ES2 LPAR2KO cells expressed elevated levels of p-AKT/AKT ratio in both the 2D and 3D forms. OVCAR3 LPAR6KO had low p-AKT/AKT ratio when cultured in 2D, but high ratio in the 3D form. LPAR KO further inhibited ovarian cancer invasion and motility.Our results demonstrate, for the first time, significant changes in LPARs' mRNA levels and Autotaxin protein levels with changes in anatomic sites of the disease that correlate to clinic-pathological parameters. Our study identifies a specific molecular machinery triggered by LPA and its' receptors that modulate tumor and can serve as therapeutic targets in this malignancy.

Citation Format: Hadil Onallah, Claes G Trope, Thea E. Hetland Falkenthal, Ben Davidson, Reuven Reich. Activity and clinical relevance of autotaxin and lysophosphatidic acid pathways in high-grade serous carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5182.