bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1449: A loss-of-function shRNA library screen for the cell invasion-suppressing genes in LNCaP prostate cancer cells
Authors: Su Bing , Baranowski Catherine , Gelman Irwin H.
Published: June 2012

Metastasis remains the primary cause of cancer-related death. Metastasis reduces the 5-year survival rate dramatically, as observed in prostate cancer, and yet, little is known about the genes that control the metastatic process. shRNA libraries are emerging as powerful tools for the identification of genes with specific functions. Here, we describe the successful application of this functional gene-discovery system by identifying genes suppressing tumor invasion, a process that is essential for metastasis. After infecting human prostate cancer cells, LNCaP, with Decode lentiviruses encoding the entire human genomic shRNA library, highly invasive variants were selected using Matrigel invasion assays. After three cycles of selection, we isolated highly invasive clones from 7 shRNA library pools. Using DNA sequence analysis and a database search, we identified five genes previously not associated with control of tumor cell invasiveness: forkhead box O4 (FOXO4), kinesin family member 3B (KIF 3B), signal transducing adaptor molecule (SH3 domain and ITAM motif) 1 (STAM), SUMO1/sentrin specific peptidase 1 (SENP1), and Homo sapiens solute carrier family 17 (SLC17A4). Silencing of FOXO4, a member of Forkhead transcription factors family, by small interfering RNAs indeed increased invasion of LNCaP cells. Reintroducing FOXO4 decreased invasion in the highly invasive prostate cancer cell lines DU145 and CWR22v1. In order to show that increased invasiveness was not a result of a change in survival ability, apoptosis and proliferation were assessed. The results indicate that FOXO4 did not affect proliferation and apoptosis in LNCaP cells. In mammals, the FOXO family comprises four members (FOXO1, FOXO3, FOXO4 and FOXO6). Though FOXO1, 3 and 4 have previously been described as having redundant functions, it appears that FOXO4 has unique metastasis suppressor function in prostate cancer. Additionally, an analysis of web-based gene expression databanks indicates that FOXO4 typically is downregulated in metastatic lesions of breast, prostate and colon cancers when compared to primary lesions. Further characterization of these candidate genes would clarify the complex mechanisms of invasion and metastasis and might reveal new classes of therapeutic targets distinct from known metastatic genes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1449. doi:10.1158/1538-7445.AM2011-1449
Abstract 145: MicroRNA-361-3p functions as an oncogenic microRNA in human oral cancer cells
Authors: Tanaka Hiroshi , Nakashiro Koh-ichi , Oka Ryota , Nishikawa Yukiko , Goda Hiroyuki , Hamakawa Hiroyuki
Published: June 2012

MicroRNAs (miRNAs) are small non-coding double-stranded RNA with sizes of 20-25 nucleotides, and inhibit protein translation by binding the 3'-untranslated region of target mRNA. Each miRNA can regulate multiple mRNAs and each mRNA can be targeted by a number of miRNAs. In cancer, miRNAs can act as not only tumor suppressor genes but also oncogenes (OncomiR). Most recent study has demonstrated OncomiR addiction in mouse pre-B-cell lymphoma. OncomiR addiction may also provide therapeutic opportunities in human cancers such as oncogene addiction. In this study, we have attempted to identify an OncomiR in human oral cancer cells through functional screening and considered whether targeting miRNA can be possible for cancer therapy. First, we performed functional screening for OncomiR in human oral cancer cells by the use of miRCURY LNATM microRNA Knockdown Library (Exiqon). We transfected 918 locked nucleic acid (LNA) antisense oligonucleotides for specific human mature miRNAs into human oral squamous cell carcinoma cells (GFP-SAS) and salivary gland cancer cells (GFP-ACCM). After transfection for 80 hours, each cell growth was evaluated. LNA antisense oligonucleotides against microRNA-361-3p (LNA-miR-361-3p) showed a remarkable growth inhibition in both types of cells as compared with non-targeting LNA oligonucleotides. We also observed the change of cell morphology, diminution of colony size, and a number of non-adherent cells after transfection of LNA-miR-361-3p. Subsequently, we examined the knockdown effect of LNA-miR-361-3p in GFP-SAS cells by quantitative RT-PCR. Compared with control oligonucleotides, the expression of miR-361-3p was significantly reduced by 71%. These effects of LNA-miR-361-3p were not observed by transfection of DNA or RNA antisense oligonucleotides for miR-361-3p. Next, we transfected synthetic human mature miR-361-3p into GFP-SAS cells to investigate the effect of miR-361-3p overexpression. Cell growth resulted in a significant 20% increase compared with non-targeting control miRNA. Furthermore, co-transfection of LNA-miR-361-3p and its decoy oligonucleotides abrogated the growth inhibitory effect by LNA-miR-361-3p in GFP-SAS cells. These results suggest that miR-361-3p functions as an OncomiR in human oral cancer cells and LNA antisense oligonucleotides are useful and efficient for silencing miRNA. Targeting miR-361-3p with LNA antisense oligonucleotides may be a useful therapeutic approach for patients with oral cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 145. doi:10.1158/1538-7445.AM2011-145
Abstract 1459: Mining for metastasis genes in osteosarcomas from p53 +/R172H mice
Authors: Xiong Shunbin , Suh Young-Ah , Quintás-Cardama Alfonso , Lozano Guillermina
Published: June 2012

Osteosarcoma (OS) is the most frequent bone cancer in young adults. The 5-year relapse free survival rates in nonmetastatic osteosarcomas are up to 65%, while metastatic osteosarcomas are only 10-20%. Therefore, identifying genes involved in OS metastasis is important to risk-stratify patients and enable development of novel therapeutic targets. In mouse models, loss of one p53 allele causes 34% of mice to develop OS without obvious metastasis; however, mice carrying a p53 missense mutation (R172H) develop metastatic OS. To screen for gene expression differences in metastatic potential between p53+/− and p53R172H/+ osteosarcomas, we used Affymetrix Mouse Genome 430 2.0 chips and compared three p53+/− and eight p53R172H/+ primary OS tumors. Two hundred thirteen genes were identified that had a p value < 0.05 and an average fold difference of 2. Only 20 of these genes had reported gene functions that affected cell migration, invasion, cell-cell junctions, or tumor phenotypes, or were selected based not only on statistical significance but also because they were consistently up (or down) regulated in all 8 R172H/+ tumors analyzed. Of these 20 genes, eight were validated by q-PCR in a larger group of tumors; S100A8, S100A9, PLA2G16, Plac8, and MAP kinase-interacting serine/threonine kinase 2 (Mnk2) were elevated while FAT4, FAT3, and Brdw1 showed decreased expression in mutant p53 tumors. To further characterize the functions of these genes in OS metastasis, we chose a metastatic OS cell line (H318) derived from p53R172H/+ tumor that had lost the wild type p53 allele for short hairpin RNAs (shRNAs) knock-down experiments followed by screening for migration and invasion. Three of these genes, PLA2G16, Plac8, and Mnk2, showed effects on migration and invasion in vitro. Further studies led us to focus on PLAG2G16. Knock-down of PLA2G16 showed decreased migration, invasion and proliferation. Additionally, we also subcutaneously injected PLAG2G16 knock-down cells into congenic wild type mice. Remarkably these cells showed dramatic decrease in tumor progression and metastasis. These effects were reproducible in another cell line with a different p53 mutant, 4T1. To determine whether we could make a non-metastatic cell adopt metastatic properties by simply expressing PLA2G16, we expressed FLAG-tagged PLA2G16 in two p53-null cell lines that lacked metastatic potential. These cells showed a 2-fold and 7-fold increase in invasion in culture, respectively. These preliminary data suggest that the expression of PLA2G16 in mutant p53 cell lines contributes to migration, invasion, and metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1459. doi:10.1158/1538-7445.AM2011-1459
Abstract 1462: Anti-metastasis effect of novel ALK5 inhibitor in both breast cancer cells in vitro and 4T1 xenograft mice in vivo
Authors: Son Jee , Yoon Eun , Kim Dae-Kee , Sheen Yhun Y.
Published: June 2012

We have synthesized novel ALK5 inhibitors and investigated their effects of on breast cancer cells as well as tumor bearing MMTV/cNeu transgenic mice and 4T1 orthopathologically xenograft balbC mice. The treatment of ALK5 inhibitors into breast tumor bearing MMTV/cNeu transgenic mice and orthotopically injected mice inhibited the numbers of nodules in the lung compared to that of untreated animal. The level of ß-casein in the lung of the breast tumor bearing MMTV/cNeu transgenic mice and 4T1 orthotopically xenograft mice were decreased by ALK5 inhibitor treatment. Zymogragh showed that the levels of MMP2 and MMP9 were decreased in the lung and breast tumor of ALK5 inhibitors treated breast tumor bearing MMTV/cNeu transgenic mice and 4T1 orthotopically xenograft mice. The action mechanisms of new ALK5 inhibitors were investigated in both breast cancer cells and breast cancer mice model. TGFß signaling, phosphorylated smad2/3 based from the western blot analysis in vitro and in vivo as well as confocal microscopic analysis in vitro was decreased by ALK5 inhibitors. And also, new ALK5 inhibitors inhibited the TGFß action on wound healing and cell invasion based on wound healing assay and invasion assay. This data strongly argue that ALK5 inhibitors have anti-metastasis effects via inhibiting ALK5 activity in vivo and in vitro. In order to gain further insight into the mechanism of ALK5 inhibitor, we have carried out experiment to examine the effects of ALK5 inhibitor in human breast cancer MDA-MB-231 cell. TGFß stimulated mesenchymal markers of epithelial mesenchymal transition (EMT), such as Snail, TWIST, N-cadherin and these were inhibited by ALK5 inhibitor concomitant treatment. Also, TGFß increased tumor invasion markers such as MMP2, and MMP9 which were inhibited by ALK5 inhibitor concomitant treatment. In summary, ALK5 inhibitors have anti-metastatic effect on human breast cancer cells as well as tumor bearing MMTV/cNeu transgenic mice.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1462. doi:10.1158/1538-7445.AM2011-1462
Abstract 1469: Mechanisms of oncostatin M (OSM)-mediated breast cancer metastasis to bone: Evidence for a role in osteoclastogenesis
Authors: Bolin Celeste M. , Tawara Ken , Sutherland Caleb , Anderson Robin , Jorcyk Cheryl
Published: June 2012

Oncostatin M (OSM) is an interleukin-6 (IL-6)-family cytokine that has been implicated in a number of biological processes including the induction of inflammation and modulation of the extracellular matrix. OSM was initially shown to inhibit the proliferation of breast cancer cell lines in vitro and has been evaluated as a potential cancer therapy. However, evidence in the literature and our data suggest that OSM may actually promote tumor invasion and metastasis. Using the orthotopic 4T1.2 mouse mammary model of metastasis, we have investigated the ability of OSM to enhance the formation of metastases in organs such as lung and bone in vivo. OSM expression was knocked down using shRNA in 4T1.2 cells, which endogenously express both OSM and its receptor. These 4T1.2-OSM cells were orthotopically injected into Balb/c mice and resulted in a 98% decrease in metastasis to bone and a 55% decrease in metastasis to lung compared to that of 4T1.2-Lac Z control cells. Metastases were quantified using qPCR to detect the vector neomycin resistance gene in post-mortem organ samples. In this study we investigate the specific cellular mechanisms that might be important for tumor cell-derived OSM promotion of metastasis to bone, such as hypoxia-inducible factor 1 alpha (HIF1α) and vascular endothelial growth factor (VEGF). An in vitro model was developed using the pre-osteoclast cell line (RAW 264.7) co-cultured with 4T1.2 mouse mammary tumor cells treated with and without recombinant OSM. Osteoclastogenesis of co-cultured RAW 264.7 cells was measured by staining for differentiated osteoclasts using the TRAP (tartrate-resistant acid phosphatase) assay and was found to increase with OSM treatment. Additionally, suppression of HIF1α expression by siRNA resulted in a 76% decrease in OSM-induced osteoclastogenesis. As RAW 264.7 cells do not express the OSM receptor beta (OSMRβ) necessary for OSM signaling, we suggest that this inhibition of osteoclast differentiation is via an autocrine signaling mechanism in the 4T1.2 cells that initiates the secretion of osteoclast differentiation factors including VEGF. Currently, we are investigating what these secreted factors from 4T1.2 cells are that affect RAW 264.7 cell differentiation and activity. Our results suggest that OSM might be an important therapeutic target in the prevention of metastatic breast cancer invasion of bone. Funded by ACS RSG-09-276-01-CSM, Susan G Komen for the Cure KG100513, and NIH/NCRR P20RR016454.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1469. doi:10.1158/1538-7445.AM2011-1469
Abstract 1475: Role of FcγRIIB in metastatic melanoma and rhabdomyosarcoma
Authors: Lu Andrew J. , Yu Yanlin , Merlino Glenn
Published: June 2012

FcγRIIB, known as immunoglobulin gamma Fc region receptor II-b, is a low affinity surface receptor protein for IgG in hematopoietic cells. As an inhibitory receptor, FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in their cytoplasmic tails that transduces an inhibitory signal on coligation with the activating receptors. FcγRIIIb is anchored to the plasma membrane via a C-terminus-linked GPI moiety. Present exclusively on human neutrophils, it plays a predominant role in binding of immune complexes. Its co-aggregation with FcγRIIa activates phagocytosis, degranulation, and the respiratory burst leading to destruction of opsonized pathogens. Activation of neutrophils leads to secretion of a proteolytically cleaved soluble form of the receptor corresponding to its two extracellular domains. Soluble Fc RIIIb exerts regulatory functions by competitive inhibition of Fcγ R-dependent effector functions and via binding to the complement receptor CR3. Dysregulation of FcγRIIB can promote the susceptibility and severity of several organ-specific and systemic autoimmune diseases invivo. Studies have recently shown that FcγRIIb increased constitutively in malignant lymphocytes and upregulated ectopically melanoma. Using cDNA microarray analysis, we have found that FcγRIIb is upregulated in metastatic melanoma and rhabdomyosarcoma (RMS). These results suggest that FcγRIIb maybe involved in regulation of progression to metastatic state in tumor.To study the role of FcγRIIb in metastatic diseases; we established model systems which stably express wild-type and mutants FcγRIIb in melanoma B16 and RMS772 cells. Cell colonies stably expressing FcγRIIb were selected and tested in vitro and in vivo. Our data showed that expression of FcγRIIb inhibited cell proliferation and caused a noticeable change in cell morphology, but did not affect cell motility and invasiveness. However, overexpression of FcγRIIb significantly stimulated lung metastasis of both melanoma and RMS cell lines. Our data suggest that over-expression of FcγRIIb promotes metastasis in melanoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1475. doi:10.1158/1538-7445.AM2011-1475
Abstract 1477: Tumor cell-leukocyte fusion in human cancer: A melanoma brain metastasis with a donor-patient hybrid genome following allogeneic BMT
Authors: Lazova Rossitza , Laberge Greggory S. , Duvall Eric , Spoelstra Nicole , Sznol Mario , Cooper Dennis , Spritz Richard , Pawelek John M.
Published: June 2012

Tumor cell fusion with motile bone marrow-derived cells (BMDCs) has long been proposed as a mechanism for metastasis, but while such fusion has been demonstrated in animal models little is known of this in human cancer. Here we report the first genetic evidence for BMDC-tumor fusion and genomic hybridization in a human melanoma brain metastasis from a patient with a prior allogeneic bone marrow transplant (male-male, sibling). STR analyses of DNA from melanoma cells throughout the tumor revealed the presence of both donor and patient alleles, with common aberrant allelic patterns at multiple chromosomal loci indicating a clonal origin of the tumor. Methods. All procedures were in accordance with HIPAA regulations and approved by the Yale HIC. Donor and patient DNAs were obtained from frozen lymphocytes and from a patient buccal swab. Formalin-fixed, paraffin-embedded tumor specimens were stained by immuniohistochemistry with common leucocyte antigen (CLA) in order to avoid leucocytes during microdissection of tumor cells. LCA-negative tumor cells were isolated with an Arturus XT laser dissection microscope system. DNA was extracted and analyzed for donor and patient alleles using forensic STR analyses at 13 chromosomal loci and amelogenin for the X and Y. Tissue sections were analyzed from 14 areas throughout the tumor. Results. DNA from donor and patient lymphocytes showed balanced diploidy at all loci and the allelic patterns were consistent with the sibling relationship. On the other hand, DNA from tumor cells contained a mixture of donor and host alleles at some loci, only donor or patient alleles at others, and numerous allelic imbalances throughout–particularly for the X and Y (X:Y = 2.8:1) that pointed to a probable tumor genotype of XXXY. With few exceptions, aberrant genetic profiles seen in one tumor section were seen in all, demonstrating a clonal origin of the tumor. Conclusions. The results provide the first genetic proof of leucocyte-tumor fusion and genomic hybridization in human cancer and indicate that the tumor was generated from a single fusion event. While this report concerns only one case, we demonstrate that forensic genetic analyses of tumors arising as secondary malignancies represents a fruitful area for research in tumor cell fusion in human cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1477. doi:10.1158/1538-7445.AM2011-1477
Abstract 148: Targeting DNA repair genes XRCC2 and XRCC4 with artificial miRNA sensitizes human brain tumor cells to radiation
Authors: Zheng Zhiming , Ng Wooi Loon , Wang Ya
Published: June 2012

Background: Previously, we reported that targeting DNA repair genes with miR-101 could sensitize human brain tumor cells to radiation (PLoS One 5: e11397, 2010). However, some tumor cell lines with high levels of endogenous miR-101 showed less response to exogenous miR-101 (our unpublished data). To overcome the resistance of the tumors to radiation, we designed artificial miRNAs to target XRCC2 (an important factor for homologous recombination repair (HRR)) and XRCC4 (an essential factor for non-homologous end-joining (NHEJ)) because both HRR and NHEJ are required for repairing radiation-induced DNA double strand breaks (DSBs) in mammalian cells and DNA DSBs are the severest threat to cell survival.

Methods: We constructed pLenti6/V5-GW/Em-GFP-miR lentiviral expression plasmids expressing artificial XRCC4 and XRCC2 miRNAs using Gateway Technology and chained artificial XRCC4 and XRCC2 miRNAs. To avoid triggering miRNA tailing and 3′-to-5′ trimming due to an extensive complementarity between a target RNA and an Argonaute1-bound miRNA, we mismatched 7 bases in the central bulge of the artificial miRNA. U87MG cells were transfected with the vector encoding both of the artificial miRNAs. XRCC4 and XRCC2 levels were measured by real-time PCR and Western blot. The effects of artificial miRNA on sensitizing U87MG cells to radiation were determined by clonogenic assay.

Results: The designed artificial XRCC4/XRCC2-miRNA could be over-expressed in U87MG cells and efficiently down-regulate the XRCC4 and XRCC2 levels. Most importantly, the artificial XRCC4/XRCC2-miRNA could efficiently sensitize the brain tumor cells to radiation.

Conclusion: The designed artificial miRNA targeting XRCC4 and XRCC2 could be a good tool to sensitize brain tumor cells to radiation, which provide a new approach for potentially improving radiotherapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 148. doi:10.1158/1538-7445.AM2011-148
Abstract 1489: Function of Rho kinases on HCC tumor growth and metastasis
Authors: Wu Ying , Whalen Pamela M. , Wang Jean Yu , Li Gary , Pocalyko David , Wang Yuli
Published: June 2012

The Rho/ROCK (Rho-associated protein kinase) pathway has been found to be frequently upregulated in hepatocellular carcinoma (HCC). Clinico-pathological research has shown that increased RhoA and ROCK2 expression are associated with the presence of tumor microsatellite formation. RhoA is over expressed in 69% of HCC, and high RhoA levels are correlated with poor survival. ROCK2 protein level is upregulated in 54% of HCC, and its expression is associated with intrahepatic metastasis. The ROCK2 knockdown by short hairpin (sh)RNA leads to significant reduction of tumor formation. These observations suggest that Rho/ROCK pathway could be a potential therapeutic target for the treatment of liver cancer. Down-regulation of the Rho/ROCK pathway may inhibit tumor metastasis. However, the role of ROCK1 in HCC metastasis is still not clear. In order to dissect out the roles of ROCK1 and ROCK2 in liver tumor formation and metastasis, we used the shRNAmir- lentiviral system to knockdown ROCK1, ROCK2, and ROCK1/ROCK2 in HCC cell lines. The RNA and protein levels of ROCK1 and ROCK2 were significantly reduced in HCC cell lines that expressed shRNAs targeting ROCK1 and ROCK2. Orthotopically implanted HCC cells carrying shRNA targeted at ROCK1 and ROCK2 either alone or in combination were examined for tumor induction in mice. Our data showed that tumor growth was significantly reduced in those mice inoculated with cells expressing shRNAs. In addition, fewer tumor nodules as compared to the control (Non-target shRNA control) group were seen in those mice. Using qRT-PCR, we confirmed that the decreased ROCK activity was associated with the reduction of ROCK1 and ROCK2 RNA levels in mouse liver samples. Conclusion: our results indicate that both ROCK1 and ROCK2 are important for HCC tumor growth. Moreover, we demonstrated that ROCK1 played a more significant role than ROCK2 in HCC intrahepatic metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1489. doi:10.1158/1538-7445.AM2011-1489
Abstract 1495: Tyrosine phosphorylation of galectin-3 regulated its cleavage by PSA
Authors: Balan Vitaly , Nangia-Makker Pratima , Raz Avraham
Published: June 2012

Galectin-3, one of 15 animal lectins, is an intracellular and extracellular lectin which interacts with intracellular glycoproteins, cell surface molecules and extracellular matrix proteins. Recent research revealed that galectin-3 (gal-3) is involved in various biological phenomena including cell growth, adhesion, angiogenesis, apoptosis, motility and metastasis. This protein is widely expressed in various tumor cells and its expression is correlated with tumorigenesis, tumor progression and metastasis. The functions of gal-3 are dependent on its binding partners, its localization and can change as a result of post-translational modifications such as cleavage and phosphorylation. Recently we demonstrated that gal-3 can be phosphorylated by c-Abl at Tyr 79, 107 and 118; Tyr107 is the major phosphorylation site. In a recent report Saraswati et al revealed that gal-3 can be cleaved by prostate specific antigen after Tyr107. Our research confirms their data and demonstrates that phosphorylation of Tyr107 blocks this cleavage. This cleavage removes the collagen like sequence from carbohydrate binding domain of gal-3 and reduces the ability of gal-3 to create oligomers, thus blocking its ability to create lattice and promote cancer behavior of the cells. These results indicate an important role of post-translational modifications of galectin-3 in prostate cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1495. doi:10.1158/1538-7445.AM2011-1495
Abstract 1497: Matrix metalloproteinase-9 mediates growth, invasion, and metastasis of human breast cancer cells
Authors: Mehner Christine , Miller Erin , Radisky Derek C. , Radisky Evette S.
Published: June 2012

Most breast cancer deaths are the consequence of metastasis from the primary site. In order to metastasize, tumor cells must escape the physical barriers that contain the growing primary tumor, enter the vascular system, exit the vascular system at a distant point, and colonize a distant organ. An important mediator of metastasis is matrix metalloproteinase-9, a member of the MMP family that has been found to be upregulated both in breast cancer cells and in the surrounding tumor stroma. Studies using animal models have sometimes produced apparently contradictory findings, due in part to methodology in which MMP-9 expression is suppressed throughout the host organism and throughout the course of tumor initiation and progression. To define the specific role of tumor cell-produced MMP-9 in metastasis of human breast cancer, we implemented a bioluminescent imaging animal model of metastasis in which luciferase-expressing MDA-MB-231 cells, infected either with control nontarget lentivirus (MDA-MB-231-NT) or with shRNA lentivirus targeting MMP-9 (MDA-MB-231-MMP9-KD), were orthotopically implanted into the mammary fat pads of immunocompromised mice. Tumor growth and development of lung metastases were assessed over 10 weeks using an IVIS imager. At 11 weeks mice were sacrificed, and tumor progression was assessed by ex vivo lung imaging and by immunohistochemical processing of the tumors and the lungs. Imaging studies revealed that primary tumors developing from MMP-9 knockdown cells grew more slowly throughout the course of the study (ranging from 25% to 50%), and that while the majority of the control group developed lung metastases, none of the mice injected with MDA-MB-231-MMP9-KD cells showed metastatic progression. After sacrifice, the measured tumor weight showed a median of 99 mg for the MDA-MB-231-MMP9-KD and 158 mg for the MDA-MB-231-NT tumors, showing that knockdown of MMP-9 inhibited tumor growth. Ex vivo imaging of the lungs confirmed the in vivo studies: no metastases were detected from MDA-MB-231-MMP9-KD cells, while all lungs of the MDA-MB-231-NT injected mice had detectable metastases (p=0.0079). Similarly, a single level immunohistochemical correlate found no metastases in the MDA-MB-231-MMP9-KD mice, while 3 of 4 mice with the non-target control cells showed large metastases (median 7×103 μm2). Our results demonstrate that expression of MMP-9 by human breast cancer cells plays a significant role in the progression of tumor growth and metastasis and provide insight into the potential of MMP-9 as a target for therapeutic intervention.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1497. doi:10.1158/1538-7445.AM2011-1497
Abstract 1499: MT1-MMP two-step activation in tumorigenesis
Authors: Golubkov Vladislav , Cieplak Piotr , Ratnikov Boris , Aleshin Alexander , Zhu Wenhong , Motamedchaboki Khatereh , Strongin Alex
Published: June 2012

Elevated activity of membrane type-1 matrix metalloproteinase (MT1-MMP) promotes cancer cell migration, invasion, and metastasis. MT1-MMP is synthesized as a zymogen, the latency of which is maintained by its prodomain. Excision by furin was considered sufficient for the prodomain release and MT1-MMP activation. We determined, however, that the full-length intact prodomain released by furin alone is a potent autoinhibitor of MT1-MMP. Additional MMP cleavages within the prodomain sequence are required to release the MT1-MMP enzyme activity. Using mutagenesis of the prodomain sequence and mass spectrometry analysis of the prodomain fragments, we demonstrated that the intracellular intradomain cleavage of the PGD-L50 site initiates the MT1-MMP activation (first step), whereas the 108RRKR111-Y112 cleavage by furin completes the removal and the degradation of the autoinhibitory prodomain and the liberation of the functional activity of the emerging enzyme of MT1-MMP (second step). Here we used the prodomain-based fluorescent biosensors to demonstrate that the proteolytic steps in MT1-MMP activation are differentially regulated in cancer cells versus normal cells. We determined that the intracellular cleavage of the PGD-L50 site represents an “on” and “off” mechanism that discriminates cancer cells from normal cells. To evaluate the significance of the PGD-L50 site cleavage in tumorigenic potential of MT1-MMP, the MCF7 cells transfected with the wild type MT1-MMP (MCF7-MT1) and the uncleavable L50D mutant (MCF7-L50D) were inoculated in the breast fat pads of nude mice. In 4 weeks palpable tumors were evident in all of the mice that received MCF7-MT1 and MCF7-L50D cells. The MCF7-L50D cells formed small tumors that reached ∼50 mm3 volume and remained unchanged from week 4 till week 10. On the contrary, the MCF7-MT1 tumors grew rapidly and reached ∼700 mm3 volume in 10 weeks. These data suggest that the intracellular cleavage of PGD-L50 site is critical for MT1-MMP two-step activation and tumorigenic activity. This intracellular cleavage does not occur in normal cells and the enzyme remained inhibited by it's non-covalently associated prodomain.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1499. doi:10.1158/1538-7445.AM2011-1499
Abstract 1502: Increased expression of KLK7 and KLK10 in papillary thyroid cancer
Authors: MacEwan Melanie , Mehta Vikas , Darr E. Ashley , Xie Suqing , Nowicki Theodore S. , Iacob Codrin , Suslina Nina , Reyes Ismael , Schantz Stimson , Shin Edward , Tiwari Raj , Geliebter Jan
Published: June 2012

The American Cancer Society estimates that there will be approximately 44,670 new cases of thyroid cancer and 1,690 deaths due to the disease in the United States in 2010. Approximately 80% of these cancers are papillary thyroid carcinoma (PTC), representing the most common endocrine malignancy. Kallikreins are serine proteases that have many physiological functions, including the remodeling of extracellular matrix (ECM). As PTC has the ability to degrade and remodel the ECM to facilitate penetration through the thyroid capsule and invasion of extra-thyroidal tissues, we investigated the expression of kallikrein 7 (KLK7) and kallikrein 10 (KLK10) in PTC. RNA expression of KLK7 and KLK10 in PTC was measured using real-time RT-PCR (normalized to GAPDH) (n = 26) and found to be significantly upregulated, with a mean fold change of 28.8 and 27.8 (P<0.05), in the tumor samples, relative to matched, normal tissue. Immunohistochemical analysis for KLK7 and KLK10 on patient samples (n = 16) revealed significantly increased immunohistochemical scoring/expression, with a mean increase of 3.0 and 2.7 (scale of 1-4; p< 0.001), respectively, in cancerous tissues, compared to adjacent, normal tissue on the same section. The data revealed a significant correlation between the expression of KLK7 and KLK10 on the both RNA level (Spearman r = 0.63, P<0.01), and on the protein level (Spearman r = 0.687, p<0.01). This coordinate expression suggests that the two kallikreins may share similar regulatory sequences. These data indicate that KLK7 and KLK10 may be useful biomarkers for the diagnosis of PTC in thyroid cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1502. doi:10.1158/1538-7445.AM2011-1502
Abstract 1504: Characterization of the murine oral squamous cell carcinoma cell line AT84 stably overexpressing uPAR
Authors: Magnussen Synnove , Hanes Robert , Hadler-Olsen Elin , Uhlin-Hansen Lars , Svineng Gunbjorg
Published: June 2012

Cancers of the oral cavity are associated with poor prognosis, and almost half of the patients diagnosed with oral squamous cell carcinoma (SCC) will die from the disease. Compared to its counterpart in skin, one main differing feature is the ability of oral SCC to metastasize. A prerequisite for metastasis is the capability to invade the surrounding tissue and penetrate into the nearest blood- or lymphatic vessel. This initial step in metastasis requires several extracellular matrix degrading enzymes. One such enzyme is uPA (urokinase-type plasminogen activator), involved in pericellular proteolysis, where high levels are correlated with poor prognosis in several cancers. Upon binding of pro-uPA to its extracellular receptor uPAR, the enzyme is cleaved and activated. uPA further activates matrix metalloproteases (MMP's), also needed for matrix degradation. In addition, ample evidence shows that binding of uPA to uPAR triggers receptor-mediated cellular signaling. uPAR lacks a membrane spanning domain hence signal transduction has to be mediated through one or several membrane partners. uPARs role in the regulation of cell adhesion, migration and proliferation has been linked to its interplay with membrane partners such as integrins, EGFR, caveolin and G-protein coupled receptors (e.g. FPRL1). To better understand the role of uPAR in the invasive growth and metastasis of oral SCCs, the mouse oral SCC cell line, AT84 [1, 2], was stably transfected with uPAR. Overexpression of uPAR resulted in down regulation of epithelial marker E-cadherin and the cells display a more mesenchymal appearance, indicating epithelial-mesenchymal transition (EMT). In addition, elevation of uPAR expression increased migration in a wound healing assay. In contrast to previous reports, an increase in MMP-9 activity was not observed in these cells, although an increase in overall proteolytic activity could be found using gelatin as substrate. The cells were reintroduced into the C3H mouse to observe the role of uPAR in cancer progression in vivo. Results from the analysis of the host-tumor interactions within this syngenous mouse model will be presented.

1. Lou, E., et al., Clinical and pathological features of the murine AT-84 orthotopic model of oral cancer. Oral Dis, 2003. 9(6): p. 305-12.

2. Schultz-Hector, S. and S. Haghayegh, Beta-fibroblast growth factor expression in human and murine squamous cell carcinomas and its relationship to regional endothelial cell proliferation. Cancer Res, 1993. 53(6): p. 1444-9.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1504. doi:10.1158/1538-7445.AM2011-1504
Abstract 1506: Up-regulation of stromal matrilin-3 expression in advanced stage high-grade serous ovarian cancer
Authors: Yeung Zachary P. , Thompson Melissa S. , Yeung Tsz L. , Zaid Tarrik M. , Wong Kwong K. , Mok Samuel C.
Published: June 2012

Objective: Transcriptome profiling performed on RNA isolated from microdissected epithelial and stromal fibroblastic components of advanced stage high-grade serous ovarian cancer (HGSC) and normal ovarian epithelial tissue identified a set of genes coding for secretory proteins that were significantly up-regulated in ovarian cancer associated fibroblasts (CAFs). One of the most significant differentially expressed genes is matrilin-3 (MATN3), which has been shown to play an important role in cartilage morphogenesis. The purpose of this study is to validate the differential expression patterns of MATN3 and its role in ovarian cancer progression.

Methods: Validation studies on MATN3 expression were carried out using quantitative RT-PCR analysis on RNA samples isolated from the microdissected stromal and epithelial components of 44 HGSC samples and 10 normal ovaries. In addition, immunohistochemistry (IHC) was performed to determine over-expression of MATN3 in the stromal component of HGSCs. To assess the functional role of matrilin-3 in ovarian cancer pathogenesis, multiple ovarian cancer cells including SKOV3 ipluc, OVCA 432, OVCA 433, and ALST were treated with exogenous recombinant MATN3. Its effect on cell proliferation was evaluated by the WST-1 assay. Furthermore, the effect of MATN3 on invasion potential was determined by counting the number of cells migrating through a transwell membrane coated with a 0.1% type I collagen matrix containing 7.5 nM MATN3.

Results: Quantitative RT-PCR analysis showed significantly over-expression of MATN3 in the stromal component of HGSC compared to the epithelial component and the normal ovaries (P=0.003). Immunohistochemistry (IHC) confirmed overexpression of MATN3 in a majority of ovarian cancer stromal specimens. When treated with recombinant exogenous matrilin-3, ALST and SKOV3 ip luc ovarian cancer cells showed a dose-dependent inhibition of proliferation by MATN3. Treatment of ovarian cancer cells SKOV3 and OVCA 432 with exogenous MATN3 significantly decreased the number of cells migrating through the transwell membrane (P<0.001).

Conclusions: Over-expression of MATN3 in ovarian cancer stroma inhibits cell proliferation and invasion potential. Further evaluating the underlying molecular mechanism of MATN3 in ovarian cancer progression is warranted.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1506. doi:10.1158/1538-7445.AM2011-1506
Abstract 1508: SLPI enhances the expression of PLSCR1 in the HEY-A8 ovarian cancer cell line
Authors: Altwerger Gary H. , Han Jasmine J. , Kohn Elise C. , Yu Minshu
Published: June 2012

Secretory leukocyte protease inhibitor (SLPI) is a secreted protease inhibitor that governs inflammation and whose importance has been demonstrated in both pathologic and non-pathologic states. We have defined SLPI as an influential component of the tumor microenvironment promoting growth, survival, and tumor aggressiveness in ovarian cancer. Extracellular SLPI interacts with progranulin, an ovarian cancer growth factor and parental molecule of the pro-inflammatory granulins. Recently, intracellular SLPI has been shown to bind to the cytosolic domain of phospholipid scramblase 1 (PLSCR1) in CD4 T-cells. PLSCR1 is a ubiquitous transmembrane proinflammatory protein with oncogenic properties. PLSCR1 overexpression has been identified in malignant colorectal cancer and xenograft tumor models with increased metastatic and invasive potential. We hypothesized that SLPI remains cytosolic or reenters the cytosol from the tumor microenvironment where SLPI can influence its intracellular receptor, PLSCR1. Preliminary western blot data demonstrated PLSCR1 protein expression in both OVCAR8 and HEY-A8 human ovarian cancer cells with OVCAR8 expressing far less PLSCR1. OVCAR8 is also known, from previous studies, to express less SLPI than Hey-A8 cells. These data led us to investigate SLPI and PLSCR1 interactions using confocal microscopy where we stained HEY-A8 cells and found colocalization of SLPI and PLSCR1. Using HEYA8 with constitutively forced expression of SLPI and protease inhibitor null mutant Leu71PheSLPI, we examined the effect of SLPI on PLSCR1. The increased secretion of SLPI in these stably transfected cells is associated with increased PLSCR1 expression regardless of protease inhibitor activity; additional mutants are currently under examination. These data suggest a positive feedback mechanism between SLPI and its intracellular receptor, PLSCR1 in human ovarian cancer cells. Inhibition of SLPI, already credentialed as a molecular target in ovarian cancer, may be enhanced by interruption of its effects on PLSCR1.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1508. doi:10.1158/1538-7445.AM2011-1508
Abstract 1512: Hypoxic activation of FVII gene in cancer cells mediated by interaction among HIF2α, Sp1, and HDAC4
Authors: Koizume Shiro , Miyagi Etsuko , Hirahara Fumiki , Nakamura Yoshiyasu , Takano Yasuo , Ruf Wolfram , Miyagi Yohei
Published: June 2012

Blood coagulation factor VII (fVII) is a key enzyme of the extrinsic coagulation cascade that is produced predominantly by hepatocytes. Tissue factor/fVIIa complex formation on the cell surface initiates key pathogenic mechanisms in cancer, including cell motility, invasion, cell survival, and angiogenesis. We have shown that various cancer cells ectopically synthesize fVII, resulting in activation of cell motility and invasion [1]. We have also shown that fVII expression is ectopically induced in ovarian cancer cells under hypoxia, potentially leading to a major complication of cancer patients, thromboembolism via secretion of fVII-positive microparticles [2].

Hypoxia inducible factors HIF1α and HIF2α are known to form a heterodimeric complex with a constitutively expressed aryl hydrocarbon receptor nuclear translocator (ARNT). HIF-ARNT complex binds to hypoxia response elements (HREs) within gene regulatory regions, resulting in transcriptional activation. We previously found that fVII gene (FVII) is inducible under hypoxia by HIF2α dependent mechanism. Unexpectedly, chromatin immunoprecipitation (ChIP) analysis revealed that HIF2α but not HIF1α was found to bind to FVII promoter without HRE [1].

In this study, we further investigated this characteristic transcriptional activation mechanism. Reporter gene, ChIP, and RNA interference (RNAi) experiments revealed that interaction between Sp1 and HIF2α in the promoter region is essential for FVII induction. Histone acetylation is known as a major mechanism for transcriptional activation. Thus, we additionally investigated effect of histone acetylation within the promoter region on HIF2α-Sp1 mediated transcriptional activation. We unexpectedly found that unlike typical transcriptional activation mechanisms, acetylation of histones negatively regulates FVII gene induction. ChIP and RNAi, followed by real-time PCR analysis revealed that a class II histone deacetylase HDAC4 is recruited to FVII promoter under hypoxia and is a coactivator responsible for this transcriptional induction. Furthermore, immunohistochemical experiments with an ovarian cancer xenograft model revealed that fVII presents in tissues strongly expressing HIF2α. These results suggest that hypoxic activation of FVII gene in cancer cells involves novel deacetylation dependent mechanism mediated by HIF2α-Sp1 interaction.

References

[1] Koizume, S. et al. Cancer Res. 66: 9453-9460, 2006.

[2] Yokota, N. et al. Br. J. Cancer, 101: 2023-2029, 2009.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1512. doi:10.1158/1538-7445.AM2011-1512
Abstract 152: MicroRNAs in head and neck squamous cell carcinoma
Authors: Uemura Mamoru , Mydlarz Wojciech , Chang Steven , Hennessey Patrick , Sun Wenyue , Shao Chunbo , Ha Patric , Sidransky David , Califano Joseph
Published: June 2012

Background: The accumulating evidence indicates that microRNAs play a key role in tumor development and progression. The aim of this study is to assess the role and function of microRNAs in head and neck squamous cell carcinoma.

Method and Results: We investigated expression profiles of microRNA in head and neck squamous cell carcinoma by using a miRNA microarray platform. Profiles from tumor samples (n=10) and nontumor tissues (n=10) were compared. We identified 24 differentially expressed miRNAs, of which 1 was significantly downregulated, while the other 23 were upregulated in tumor tissues. Mir-21 was most notably upregulated in tumor tissues (P=0.0002), and mir-375 was a potential tumor suppressive microRNA that was significantly downregulated in tumors (P=0.0002). Expression profiles of these two microRNAs were validated by qRT-PCR with separate clinical samples (16 tumors and 15 normal tissues). In vitro assays confirmed that mir-21 stimulated cell growth and mir-375 had a tumor suppressive role. Next, to assess downstream target genes, cells were subjected to microarray analysis after transient transfection with each microRNA or its negative control. We also conducted a bioinformatics search (TargetScan, PicTar, miRanda). By combining these data, we identified potential target genes including YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide) for mir-375 and BNC2 (Basonuclin 2) for mir-21. These potential target genes were downregulated after forced expression of mir-375 or mir-21. The activities of a luciferase reporter containing these microRNA's binding sequences at the 3’ untranslated region (UTR) of some potential target mRNAs were repressed by the ectopic expression of miR-375 or miR-21, suggesting that some of these candidates are specific targets of these microRNAs.

Conclusions: Mir-21 is a potential oncogenic microRNA, and miR-375 is a potential tumor-suppressive microRNA in head and neck squamous carcinoma. Further investigation including pathway analysis of these microRNAs may lead to the detection of therapeutic targets of head and neck cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 152. doi:10.1158/1538-7445.AM2011-152