Donghong Ju

Abstract 5163: MMP3 increases expression of CXCL7.

MMP3 leads to epithelial-mesenchymal transformation (EMT) and malignant transformation of cultured cells. Previously, increased expression of the CXC chemokine CXCL7 was associated with EMT. The hypothesis tested is that MMP3 increases the expression of CXCL7 Methods: Human breast cells (MCF10AT (proliferative), MCFDCIS.com (in situ), MCFCA1a (malignant)) cultured in serum free medium were exposed to MMP-3 for 4 days, and washed 3 times with PBS, then cultured in MMP-3 free condition medium for three more days. Cells were collected every day for RNA extraction, and analyzed for relative CXCL7 gene expression using RT real time PCR. Cells sampled from day 0 were used as control to normalize the results. GADPH was control. Cells culture supernatant also collected every day for CXCL7 protein level expression. Protein concentrations were measured by enzyme-linked immunosorbent assay (ELISA) using human CXCL7/NAP-2 Duo set ELISA Development System. Measurements were done in triplicate; results are representative of three experiments. Results: When exposed to MMP-3, the CXCL7 RNA level increased MCF-10AT cells RNA level increased 1.5 fold, and there was no change in CXCL7 protein level in MCF-10AT cells. For MCFDCIS.com cells, the CXCL7 RNA level increased 2.5 fold, and CXCL7 protein level increased 1.6 fold. For MCFCA1a cells, the CXCL7 RNA level increased 2.6 fold, CXCL7 protein level increased 1.5 fold. The peak expression was 3-4 days. Removal of MMP-3 caused CXCL7 expression to return back to baseline in MCF10AT, DCIS.com and malignant Ca.cl1 cells. Conclusion: The expression of chemokine CXCL7 in breast cell (MCF10AT, DCIS.com, MCFCA1a) was up regulated at both on RNA and protein level (except for MCF10AT) when exposed to MMP-3, and removal of MMP-3 caused CXCL7 expression to return to baseline. The stromal influence by MMP3 on breast cells and breast cancer cells may direct further progression toward the malignant phenotype and provides another therapeutic pathway target. Citation Format: Donghong Ju, Mary A. Kosir. MMP3 increases expression of CXCL7. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5163. doi:10.1158/1538-7445.AM2013-5163

Challenging paradigms: long non-coding RNAs in breast ductal carcinoma in situ (DCIS)

OVERVIEW The continuing challenges to the treatment of breast ductal carcinoma in situ (DCIS) warrant a new avenue of investigation. In parallel, a startling revelation of the first post-genomic decade has been the rising prominence of long non-coding RNAs (lncRNAs) as key gene regulators, including in breast cancer. High-throughput experimental surveys of the human transcriptome show that lncRNAs are abundant. In contrast to microRNAs (miRNAs), lncRNAs possess multiple, diverse, heterogeneous molecular mechanisms of action making them high-level master regulators of gene regulatory networks and pathways in cancer. In particular, lncRNAs directly regulate the activity of transcription proteins and epigenetic modifiers, which, in turn, govern their own downstream sets of target genes. Therefore, provided that such lncRNA regulatory activities are functional in cancer, including DCIS, targeting the lncRNAs or the regulatory networks which they mediate can represent an important new avenue of treatment. Development of lncRNAtargeting therapies may emerge into a key innovation generated in clinical and translational research by the innovative use of the latest human genome annotation datasets combined with high throughput transcriptome analyses. We propose a paradigm shift: direct interrogation of lncRNAs in the molecular characterization of DCIS subtypes, including the idea that lncRNAs may be the trigger for progression from in situ to invasive cancer.

Primate-specific oestrogen-responsive long non-coding RNAs regulate proliferation and viability of human breast cancer cells.

Long non-coding RNAs (lncRNAs) are transcripts of a recently discovered class of genes which do not code for proteins. LncRNA genes are approximately as numerous as protein-coding genes in the human genome. However, comparatively little remains known about lncRNA functions. We globally interrogated changes in the lncRNA transcriptome of oestrogen receptor positive human breast cancer cells following treatment with oestrogen, and identified 127 oestrogen-responsive lncRNAs. Consistent with the emerging evidence that most human lncRNA genes lack homologues outside of primates, our evolutionary analysis revealed primate-specific lncRNAs downstream of oestrogen signalling. We demonstrate, using multiple functional assays to probe gain- and loss-of-function phenotypes in two oestrogen receptor positive human breast cancer cell lines, that two primate-specific oestrogen-responsive lncRNAs identified in this study (the oestrogen-repressed lncRNA BC041455, which reduces cell viability, and the oestrogen-induced lncRNA CR593775, which increases cell viability) exert previously unrecognized functions in cell proliferation and growth factor signalling pathways. The results suggest that oestrogen-responsive lncRNAs are capable of altering the proliferation and viability of human breast cancer cells. No effects on cellular phenotypes were associated with control transfections. As heretofore unappreciated components of key signalling pathways in cancers, including the MAP kinase pathway, lncRNAs hence represent a novel mechanism of action for oestrogen effects on cellular proliferation and viability phenotypes. This finding warrants further investigation in basic and translational studies of breast and potentially other types of cancers, has broad relevance to lncRNAs in other nuclear hormone receptor pathways, and should facilitate exploiting and targeting these cell viability modulating lncRNAs in post-genomic therapeutics.

Strategic endothelial cell tube formation assay: Comparing extracellular matrix and growth factor reduced extracellular matrix

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patients blood stream by hijacking the process of angiogenesis, in which new blood vessels are formed from existing blood vessels. The CXCR2 (chemokine (C-X-C motif) receptor 2) receptor is a transmembrane G-protein-linked molecule found in many cells that is closely associated with angiogenesis1. Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. The tube formation assay is useful for studying angiogenesis because it is an excellent method of studying the effects that any given compound or environmental condition may have on angiogenesis. It is a simple and quick in vitro assay that generates quantifiable data and requires relatively few components. Unlike in vivo assays, it does not require animals and can be carried out in less than two days. This protocol describes a variation of the extracellular matrix supporting endothelial tube formation assay, which tests the CXCR2 receptor.

Abstract 1363: VEGF reduction facilitates angiogenesis by CXCR2

PURPOSE: With multiple pathways for angiogeneis, the purpose of the study is to quantify the expression of angiogenic tyrosine kinase receptors during endothelial tubule formation while inhibiting a G-protein linked CXCR2 receptor that binds angiogenic CXC chemokines. METHODS: Using endothelial tubule formation assays on Matrigel and Growth Factor Reduced (GFR) Matrigel, and without added VEGF, human umbilical vascular endothelial cells (HUVEC) were treated with a selective small molecule non-peptide inhibitor of CXCR2 called SB225002 (N-(2-hydroxy-4-nitrophenyl)-N9-(2-bromophenyl)urea compared with control. Previous dose-inhibition curves established optimal dosing of SB225002. After 6 hours of incubation, endothelial tubule formation was quantified by counting tubule length in four random microscopic fields using the microscope camera software NIS-Elements AR 3.10 in triplicate and compared to control. In parallel, endothelial cells were collected from the Matrigel under the same conditions for quantification of angiogenesis receptors and growth factors by RT-PCR in triplicate. RESULTS: There was significant inhibition of endothelial tubule formation on Matrigel (and GFR Matrigel) by the addition of SB225002 (40-58% of inhibition) under conditions of no added VEGF when compared to control. The expression of mRNA in HUVEC cells harvested from Matrigel was not different for VEGFR1-3, FGFR1-3, and PDGFRA,B with or without the inhibitor. Under conditions of no added VEGF, the expression of CXCR2 mRNA was the same with or without the inhibitor, but the endothelial tubule formation was significantly inhibited. CONCLUSION: Under growth factor reduced conditions, including no added VEGF, the endothelial tubule formation and angiogenenic tyrosine kinase receptor expression was similar to standard matrigel. However, when a selective non-peptide inhibitor of CXCR2 (SB225002) was added, endothelial tubule formation was inhibited without change in expression of other angiogenesis receptors from HUVEC cells retrieved from the matrigel. Therefore, tubule formation continues with the chemokine/CXCR2 axis and may be an additional pathway that circumvents other attempts at angiogenesis inhibition targeting tyrosine kinase inhibitors. Citation Format: Mary A. Kosir, Donghong Ju. VEGF reduction facilitates angiogenesis by CXCR2. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1363. doi:10.1158/1538-7445.AM2015-1363

Abstract 3008: Effects of HSPG on inhibition of chemokine-induced angiogenesis

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA PURPOSE: The purpose of the study is to inhibit angiogenesis by using using specific inhibitors to the CXCR2 receptor that binds angiogenic CXC chemokines, while varying the concentration of heparan sulfate proteoglycan (HSPG), an important molecule for multiple angiogenesis factors. METHODS: Using endothelial tubule formation assays on Matrigel, human umbilical vascular endothelial cells were treated with either antibody to CXCR2 or a small selective non-peptide inhibitor SB225002 (N-(2-hydroxy-4-nitrophenyl)-N9-(2-bromophenyl)urea at varying concentrations and compared to control creating a dose-response curve. Experiments were repeated using Growth Factor Reduced (GFR) Matrigel which has 40-50% less HSPG. After 4 hours incubation, endothelial tubule formation was quantified by counting tubule numbers in four random microscopic fields using the microscope camera software NIS-Elements AR 3.10 in triplicate and compared to control. RESULTS: There was a significant and dose-dependent inhibition of endothelial tubule formation on Matrigel by SB225002 at concentrations ranging from 0.56 µM to 11 µM (p=0.04 to 0.004). The IC50 was 5.2 µM. There was also significant inhibition of tubule formation with anti-CXCR2 antibody at 20-40 µg/ml in a dose-dependent manner (p=0.001 and 0.00003), with IC50 of 33.6 µg/ml on Matrigel. However, on GFR Matrigel, the IC50 for SB225002 inhibition of endothelial tubule formation was lower at 0.61 µM with a significant dose-dependent inhibition from 0.56 µM to 11 µM (p=0.0004 to 0.00001). Anti-CXCR2 antibody inhibition of tubule formation was also significant on GFR Matrigel at 20 µg/ml (p=0.006) with IC50 of 18.6 µg/ml. CONCLUSION: A selective non-peptide inhibitor of CXCR2 (SB225002) inhibited endothelial tubule formation with 8.5 times a lower dose when HSPG concentrations were reduced using GFR Matrigel. Angiogenesis factors such as VEGFA, FGF-2 and CXC chemokines require HSPGs on the cell surface of endothelial cells, so optimal control of angiogenesis may require control of HSPGs as well. The inhibition of transmembrane CXCR2 is important for emerging therapies for inflammation and malignancy, and associated angiogenesis. Citation Format: Donghong Ju, Cecelia Speyer, David Gorski, Mary A. Kosir. Effects of HSPG on inhibition of chemokine-induced angiogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3008. doi:10.1158/1538-7445.AM2014-3008

Abstract 1842: Identifying long noncoding RNAs in breast cancer microarrays.

Long noncoding RNAs (lncRNA) are powerful regulators of gene expression. The emerging role of long noncoding RNAs (lncRNA) is facilitated by the development of new technologies such as tilling array and next generation sequencing. In this study, our research focused on lncRNA9s regulatory mechanisms in breast cancer. Methods: The Gencode v7 lncRNA and FANTOM5 lncRNA datasets were integrated as our lncRNA transcriptome. The expression profile was from four publications of microarray analysis in breast cancer. First, we checked the Affymetrix probe dataset U133 plus with our lncRNA transcriptome. If a probe was on the same strand and 100% fell into the exonic region of one lncRNA transcript, then the probe could this lncRNA. We integrated all lncRNAs from our transcriptome which matched the probes from the supplementary data of four papers and selected those occurred more than once. Results: There were 23 nonredundant lncRNA candidates identified. Among those 23 candidates, PTENP1 was identified. PTENP1 is the pseudogene of tumor suppressor gene PTEN. The 3’UTR can bind the same miRNAs as the related protein-coding gene, PTEN. This suggests that pseudogenes may have a biological function as ‘decoys’, sequestering miRNAs and thereby affecting their regulation of expressed genes. Another candidate identified was ENST00000415285.1, which is the antisense gene of FGF14. FGF14 is a member of the fibroblast growth factor (FGF) family. FGF family members possess broad mitogenic and cell survival activities, and are involved in a variety of biological processes, including tumor growth and invasion. ENST00000415285.1 is up-regulated in breast cancer, while the expression level of FGF14 is unknown, so further research is necessary to predict the possible mechanism of regulation to FGF14. Conclusion: LncRNAs are identified in datasets for breast cancer that may elucidate further regulatory mechanisms driving the pathogenesis of breast cancer. Citation Format: Hui Jia, Leonard Lipovich, Donghong Ju, Mary A. Kosir. Identifying long noncoding RNAs in breast cancer microarrays. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1842. doi:10.1158/1538-7445.AM2013-1842

Abstract 307: Acquisition of the invasive phenotype and EMT profile by CXCL7 transfection

Introduction: The expression of CXCL7 is associated with the invasive phenotype through interaction with the CXCL7/CXCR2 axis and heparan sulfate. A component of the epithelial to mesenchymal transition (EMT) is degradation of extracellular matrix and acquisition of migration, both of which support invasiveness. The purpose of this study is to quantify the expression of genes associated with EMT by CXCL7-expressing cells with the invasive phenotype. Methods: Total RNA from CXCL7-transfected breast cells (MCF10AT) was tested for relative gene expression using RT 2 Profiler TM PCR array for EMT (SABiosciences). Vector transfected cells were compared. Each result was verified by real time PCR assay. Results: The expression of GNG11 and TCF4 was upregulated and SPP1 downregulated as expected for the EMT profile. Specifically, GNG11 (Guanine nucleotide binding protein (G protein), gamma 11) expression was increased 2.94 fold and TCF4 (Transcription factor 4) increased 1.89 fold. The expression of SPP1 (Secreted phosphoprotein 1) was decreased by 6.8 fold. The expression of ZEB2 (Zinc finger E-box binding homeobox 2) was decreased by 1.96 as a transcription factor. Summary: The CXCL7-transfected cells with invasive phenotype express genes associated with the EMT expression profile including G-protein signaling pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 307. doi:1538-7445.AM2012-307