bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 273: Molecular markers promoting metastases to the brain via EMT: Genes, proteins, and functional analysis.
Authors: Jeevan Dhruve S. , Sridhara Sudeepta , Braun Alex , Murali Raj , Jhanwar-Uniyal Meena
Published: August 2014

Brain metastasis is the most common cause of mortality and morbidity of many cancers and occurs in more than 40% of patients. The metastatic cascade entails an orderly sequence of steps enabling tumor cells to migrate from the primary tumor and colonize at secondary locations. The underlying mechanisms of metastatic dissemination to the brain remain to be defined. However, In order to achieve this metastatic potential a cancer cell may go through a cellular reprogramming process, namely epithelial-to-mesenchymal transition (EMT), and its counterpart mesenchymal-to-epithelial transition (MET), by acquiring multiple genetic alterations and a stem cell like phenotype. We hypothesized that EMT would play a crucial role in brain metastasis. In order to achieve our goal we studied the expression of markers of EMT, MET, and stem cells in metastatic brain tumor samples. Microarray analysis of metastatic tumors was also done using a plate form of 17,000 genes. Furthermore, functional analysis establishing the metastatic phenotype of cancer cells towards a cerebral environment was also demonstrated using metastatic cell lines. Our results demonstrated that the expressions of EMT markers (Snail, TWIST, pSTAT3, NFkB), as well as the mesenchymal marker Vimentin, were present in metastatic tumor samples. The stem cell marker CD44 was also highly expressed. Gene expression analysis established the high presence of transcriptional factors associated with differentiation and reprogramming. Lastly, Immunofluorescence analysis demonstrated the presence of the mesenchymal marker Vimentin in brain tumor specimens. Tumor cells grown in astrocytic media displayed an increased cell proliferation, as well as enhanced S-phase cell cycle entry using an EDU incorporation technique. Migration of primary tumor cells was significantly enhanced in astrocytic media as evidenced by scratch wound migration. Furthermore, the tumor cells have a greater affinity for astrocytic media as demonstrated by chemotatic migratory tests. Moreover, co-culture analysis of primary tumor cells with astrocytic cells shows amicable mutual growth with Immunofluorescence demonstrating cell-to-cell interaction via E-Cadherin. In conclusion results of this study suggests that reprogramming via EMT/MET plays a crucial step in brain metastasis, and that the cerebral milieu provides a suitable microenvironment for metastatic cells to grow and disseminate.

Citation Format: Dhruve S. Jeevan, Sudeepta Sridhara, Alex Braun, Raj Murali, Meena Jhanwar-Uniyal. Molecular markers promoting metastases to the brain via EMT: Genes, proteins, and functional analysis. [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 273. doi:10.1158/1538-7445.AM2013-273
Abstract 2746: Microsomal prostaglandin E2 synthase-1 may provide a novel specific therapeutic target in neuroblastoma.
Authors: Kock Anna , Rasmuson Agnes , Korotkova Marina , Idborg Helena , Johnsen John Inge , Jakobsson Per-Johan , Kogner Per
Published: August 2014

Background: Neuroblastoma (NB) cells are enriched in the omega-6 fatty acid arachidonic acid, the substrate for the cyclooxygenase (COX) enzymes and prostaglandin biosynthesis. The inducible isoform, COX-2 is overexpressed in NB and NB cells produce prostaglandin E2 (PGE2) that acts as an autocrine and/or paracrine survival and proliferation factor. Downstream of the COX enzymes, specific synthases are responsible for the production of the respective prostaglandins. Microsomal prostaglandin E2 synthase-1 (mPGES-1) specifically converts PGH2 to PGE2 and is thought to primarily couple to COX-2. The aim of this study was to investigate if inhibition of mPGES-1 could represent an alternative therapeutic approach to COX- inhibition in NB.

Methods: Western blot and immunohistochemistry were used for protein detection. Cell viability of seven NB cell-lines treated with the mPGES-1 inhibitor CAY1052 was determined by MTT-assay. Stable mPGES-1 knockdown SK-N-BE2 clones were established using shRNA and the clonogenic capacity was analysed by clonogenic assay. To study the in vivo effect of COX inhibition, four week old homozygous TH-MYCN mice were treated with 10mg/L diclofenac for two weeks. Ex vivo analyses of COX-metabolites in tumors were performed by LC-MS/MS.

Results: Expression of mPGES-1 in human NB tumors and in NB cell lines could be detected. Inhibition of mPGES-1 reduced NB cell growth in vitro and knockdown of mPGES-1 significantly reduced the clonogenic capacity. Expression of COX-1, COX-2 and mPGES-1 in TH-MYCN tumors could be detected and treatment with the dual COX-inhibitor diclofenac significantly reduced tumour weight, compared to untreated animals. Ex vivo analysis of tumor tissues from treated animals revealed a significantly decreased level of COX metabolites compared to controls. The expression of mPGES-1was not affected by the treatment. Furthermore, cells staining positive for cleaved caspase-3 were more prevalent in treated tumors indicating apoptosis Induction.

Conclusion: We found that mPGES-1 is expressed in NB, with a potential role for PGE2 synthesis and tumor growth. mPGES-1 represents an alternative therapeutic target for inhibiting NB growth through specific PGE2 inhibition and the TH-MYCN model is well suited for in vivo studies with this purpose.

Citation Format: Anna Kock, Agnes Rasmuson, Marina Korotkova, Helena Idborg, John Inge Johnsen, Per-Johan Jakobsson, Per Kogner. Microsomal prostaglandin E2 synthase-1 may provide a novel specific therapeutic target in neuroblastoma. [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 2746. doi:10.1158/1538-7445.AM2013-2746
Abstract 2751: Wnt/β-catenin regulates the expression of MGMT through TCF/LEF transcription: new strategy for DNA alkylators in cancer therapy.
Authors: Wickström Malin , Baryawno Ninib , Dyberg Cecilia , Milosevic Jelena , Sveinbjörnsson Baldur , Northcott Paul A. , Taylor Michael , Kool Marcel , Kogner Per , Johnsen John I.
Published: August 2014

Background: O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that protects DNA from the biological effects of alkylating agents. MGMT has been implicated in resistance to alkylating drugs such as temozolomide used in the clinic against brain tumors. Temozolomide has been shown to be ineffective in tumors expressing elevated levels of MGMT. Finding agents that induce MGMT deficiency and increase the susceptibility to alkylating chemotherapeutic agents is highly warranted.

Methods: The correlation between Wnt activity and MGMT expression was investigated in primary medulloblastomas, gliomas and colon cancer using gene expression cohorts. Cell lines from medulloblastomas, gliomas, colon cancer and neuroblastomas were examined for Wnt/β-catenin activity and MGMT expression using immunoblotting and real-time RT-PCR. siRNA against β-catenin, forced overexpression of β-catenin, and MGMT promotor reporter plasmids were used to study β-catenin mediated regulation on MGMT. Cell cytotoxicity and clonogenicity of chemotherapeutic drugs in combination with celecoxib were examined in cell lines using fluorometric microculture cytotoxicity assay and clonogenic assay, respectively. In vivo, nude NMRI mice carrying xenografts were treated with celecoxib in combination with temozolomide.

Results: Wnt signaling activity was shown to be correlated to increased MGMT expression levels both in primary tumors and cell lines. Transfection experiments revealed that β-catenin regulates MGMT expression. The clinically available COX-2 inhibitor celecoxib augmented the cytotoxic effect of temozolomide in cells with elevated MGMT expression through a reduction of Wnt/β-catenin activity and a subsequent reduction in MGMT expression.

Conclusions: This study demonstrates that celecoxib reduces the transcription of the MGMT gene through a prostaglandin E2-Wnt/β-catenin route and thus increases the susceptibility to temozolomide. These finding are the first reported that Wnt/β-catenin pathway regulates the expression of MGMT and thus provides a rational approach for improved efficacy of temozolomide therapy in the treatment of medulloblastoma and other cancers.

Citation Format: Malin Wickström, Ninib Baryawno, Cecilia Dyberg, Jelena Milosevic, Baldur Sveinbjörnsson, Paul A. Northcott, Michael Taylor, Marcel Kool, Per Kogner, John I. Johnsen. Wnt/β-catenin regulates the expression of MGMT through TCF/LEF transcription: new strategy for DNA alkylators in cancer therapy. [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 2751. doi:10.1158/1538-7445.AM2013-2751
Abstract 2770: Anti-leukemic response of a NSAID, tolfenamic acid.
Authors: Sutphin Robert M. , Connelly Sarah F. , Lee Chris M. , Sankpal Umesh T. , Eslin Don , Basha Riyaz
Published: August 2014

Tolfenamic acid (TA), a non-steroidal anti-inflammatory drug is known to inhibit human cancer cells and mouse tumor growth in some cancer models; however its anti-leukemic response is not yet evaluated. Research from our laboratory and others showed that TA targets specificity protein (Sp) transcription factors which mediate the expression of several genes associated with cancer. We also showed that TA inhibits the expression of survivin, a key member of inhibitor of apoptosis family in several human cancer cells. Recent work from several laboratories revealed a strong association of survivin in leukemia. We hypothesize by targeting Sp proteins and survivin, TA can act as an anti-leukemic agent. The anti-proliferative response of TA was determined using four human leukemia cell lines, Jurkat (acute T-cell leukemia), Nalm-6 (pre-B cell leukemia), Molt-4 (acute lymphoblastic leukemia; T lymphoblast), and Reh (acute lymphoblastic leukemia, non-T; non-B) Cells were treated with increasing (25/50/75/100 μM) concentrations of TA and the cell viability was measured at 24, 48, and 72 h post-treatment using CellTiter-Glo kit. Results show a consistent decrease in cell viability in a dose and time-dependent manner. Confirmatory studies to elucidate the mechanism of action were conducted using selected cell lines, Jurkat and Nalm-6. Apoptosis and cell cycle analysis was performed using flow cytometry. The expression of c-PARP, Sp1, survivin, CDC2, CDC4, Cyclin D3 and pRb was determined by Western blot analysis and the caspases were measured by using Caspse-Glo kit(s). Results showed a significant increase in the apoptotic (annexin V positive) cell population following TA treatment, while cell cycle phase distribution analysis showed G0/G1 arrest. TA-induced cell apoptosis is supported by robust activation of caspases (3/7, 8 and 9), and the expression of c-PARP. TA down-regulated the expression of CDC2, CDC4, Cyclin D3 and pRb that mediate the early phases of cell cycle. In summary, TA modulated the expression of critical candidate genes associated with the early phases of cell cycle with validated efficacy in causing G0/G1 arrest. Western blot results reveal that TA significantly decreases Sp1 and survivin expression. Further work is needed to clarify the role of TA as a novel therapeutic agent for leukemia.

Citation Format: Robert M. Sutphin, Sarah F. Connelly, Chris M. Lee, Umesh T. Sankpal, Don Eslin, Riyaz Basha. Anti-leukemic response of a NSAID, tolfenamic acid. [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 2770. doi:10.1158/1538-7445.AM2013-2770
Abstract 2772: Inhibition of EGFR activity reduces AML cell survival via inhibition of downstream Akt and Erk signaling pathways.
Authors: Scheepers Ellen R. , Kampen Kim R. , de Bont Eveline SJM
Published: August 2014

Epidermal growth factor receptor (EGFR) inhibitor erlotinib, has been shown to induce complete remission of acute myeloid leukemia (AML) in two patients with concurrent lung cancer, suggesting a prominent role for EGFR in AML. However, EGFR expression analysis in AML is poorly defined and the role of EGFR in AML is still unclear.

In this study, we determined the EGFR protein expression in different human AML cell lines and studied the impact of EGFR inhibition by canertinib on the AML cell survival, proliferation, apoptosis and their downstream signaling routes. Using flowcytometry, we have shown that total EGFR as well as the phosphorylated residue Tyr1068 is expressed in five out of the eight AML cell lines (range of 4.9-46.6% for total and phosphorylated EGFR). EGFR phosphorylation was found to be induced by EGF stimulation, which could be prevented by canertinib treatment, showing functional activity and specificity of the EGFR phosphorylation site. Canertinib was shown to decrease the AML cell survival sufficiently (at a median IC50 of 6.2 μM, range: 0.9 - 15.2 μM canertinib in n=8 AML cell lines) as measured by WST-1 assays. The lower cell survival was the net result of lower numbers of mitotic phospho-histone H3 positive cells and an increase in the number of Annexin V positive cells. Immunoblot analysis revealed that canertinib treatment (5uM) reduced the phosphorylation of Akt and Erk intracellular proteins downstream of the EGFR signaling pathway in AML cells. Long-term canertinib treatment (5uM) showed that downstream targets were re-activated after 24 hours, which suggest kinomic reprogramming by alternative escape mechanisms compensating for the loss of EGFR activity. These findings suggest encouraging possibilities for targeting EGFR as a therapeutic strategy in AML. Future studies are necessary to determine the exact mechanism through which EGFR activity might contribute to AML progression, relapses as well as to interact with possible escape mechanisms of AML cells.

Citation Format: Ellen R. Scheepers, Kim R. Kampen, Eveline SJM de Bont. Inhibition of EGFR activity reduces AML cell survival via inhibition of downstream Akt and Erk signaling pathways. [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 2772. doi:10.1158/1538-7445.AM2013-2772
Abstract 2800: IL-5 induced migration of HT1376 cells via ERK1/2-mediated MMP-9 expression by inducing activation of NF-κB .
Authors: Lee Eo-jin , Lee Se-Jung , Moon Sung-Kwon
Published: August 2014

Interleukin-5 (IL-5) plays an important role in the growth and differentiation of human B cells and eosinophils. However, little is known about the effect of IL-5 on cancer cells. In this study, we investigated the molecular mechanism involved in the IL-5-induced migration of bladder cancer HT1376 cells. Our results indicated that IL-5 significantly enhanced migration and MMP-9 expression in HT1376 cells. We also found that IL-5 induces transcriptional activation of the binding of NF-kB and AP-1, which are two important nuclear transcription factors that are linked to MMP-9 expression in HT1376 cells. In subsequent experiments, we found activation of ERK1/2 in IL-5-treated HT1376 cells. To examine the involvement of the ERK1/2 signaling pathway on IL-5-induced cell responses, we pretreated HT1376 cells with the ERK1/2 inhibitor U0126 followed by IL-5 treatment. The results showed that U0126 treatment inhibited migration of IL-5-treated HT1376 cells. Moreover, IL-5-stimulated MMP-9 expression was suppressed by the addition of U0126. Inhibition of ERK1/2 function consistently rescued transcriptional activity of NF-kB, without altering AP-1 activation, in IL-5-treated cells. Finally, inhibition of IL-5 specific receptor IL-5R alpha by small interfering RNA (siRNA) suppressed migration, ERK1/2 activation, MMP-9 expression and binding activation of NF-kB in IL-5-treated HT1376 cells. The results of the present study indicate that the IL-5/IL-5R alpha dyad induced cell migration through ERK1/2-mediated expression of MMP-9 by binding activation of NF-kB in bladder cancer cells. In conclusion, these novel findings indicate that binding of IL-5 to IL-5R alpha plays a critical role in MMP-9 expression, which might be involved in the migration of bladder cancer.

Citation Format: Eo-jin Lee, Se-Jung Lee, Sung-Kwon Moon. IL-5 induced migration of HT1376 cells via ERK1/2-mediated MMP-9 expression by inducing activation of NF-κB . [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 2800. doi:10.1158/1538-7445.AM2013-2800
Abstract 2803: Disabeled-2 (Dab-2) modulates platelet-cancer cell interactions through its sulfatide binding domain.
Authors: Andreano Kaitlyn , Fu Xiangping , Capelluto Daniel , Finkielstein Carla
Published: August 2014

Platelets’ role in cancer progression and metastasis has largely been attributed to platelet-mediated enhancement of tumor cell survival, extravasation (exiting form capillaries and entering organs), and angiogenesis. Correlations exist between the ability of some tumor cells to aggregate platelets in vitro and their metastatic potential in vivo that it is manifested as a hypercoagulable state found in most cancer patients. We have identified a tumor suppressor molecule (Dab-2) that it is released upon platelet activation and that modulates the extent of blood clotting. Our results show Dab-2 is released from alpha-granules in the platelet to the membrane surface where specifically binds to the integrin receptor, thus inhibiting platelet aggregation. Binding of Dab-2 to integrins is modulated by sulfatides, a form of glycosphingolipid that accumulate in platelet membrane and effectively compete for Dab-2 levels. Interestingly, we found that the N-terminal region of Dab-2 specifically binds to sulfatides through two conserved sulfatide-binding sites. Upon activation, sulfatides protect Dab-2 from thrombin cleavage, facilitate its internalization and modulate the surface expression of P-selectin, a coagulation protein needed for stabilization of platelet aggregates. P-selectin also mediates both tumor cell adhesion to vascular endothelial cells and the interaction between activated platelets and cancer cells (emboli) during metastasis. Interestingly, our data show that Dab-2/sulfatide recognition influences the stability of platelet aggregates heterotypically with cancer cells through sulfatide binding and, indirectly, by controlling P-selectin levels. Thus, we hypothesize that manipulation of Dab-2 function in response of platelet activation will impact emboli formation offering an alternative route for therapeutic strategies aimed to control metastatic processes.

Citation Format: Kaitlyn Andreano, Xiangping Fu, Daniel Capelluto, Carla Finkielstein. Disabeled-2 (Dab-2) modulates platelet-cancer cell interactions through its sulfatide binding domain. [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 2803. doi:10.1158/1538-7445.AM2013-2803
Abstract 2812: Macrophage Migration Inhibitory Factor (MIF) regulates monocytic MDSC abundance to contribute to tumor growth and metastasis.
Authors: Simpson Kendra D. , Templeton Dennis J. , Cross Janet V.
Published: August 2014

The inflammatory cytokine, Macrophage Migration Inhibitory Factor (MIF) is a critical mediator of numerous inflammatory disease processes. In addition, MIF is overexpressed in many cancers and the degree of expression correlates with tumor aggressiveness. We identified MIF as a target for inhibition by sulforaphane (SFN), a cancer chemopreventive agent prevalent in broccoli. Based on this observation and the correlation between MIF expression and poor prognosis in human patients, we set out to determine how MIF might contribute to tumor growth and progression.

We hypothesized that, as an inflammatory cytokine, MIF may regulate the interaction between the tumor and the host immune response, rather than impacting an intrinsic property of the tumor cells. We are exploring these questions using the syngeneic 4T1 model of breast carcinoma. This model recapitulates many aspects of human breast tumor progression, including spontaneous metastasis from the mammary fat pad to similar target organs. We found that MIF expression in the tumor cells increases tumor growth and metastasis in an orthotopic tumor model. Importantly, the MIF dependent growth differences are only manifested in vivo, as the MIF depleted cell line is not compromised in growth or colony formation in vitro, suggesting that MIF may be contributing to the interaction between the tumor and the host immune response. This was confirmed by demonstrating that MIF depleted and MIF containing tumors exhibit similar growth and metastasis properties in immunodeficient animals. Evaluation of the immune cell infiltrates in the MIF containing and MIF deficient tumors reveal that the abundance of monocytic MDSC within the tumor is dependent on MIF expression. Finally, we demonstrate that depletion of MDSCs abolishes the impact of MIF on tumor growth, suggesting that these cells are critical for MIF to mediate its effects. We are currently exploring the mechanisms by which MIF controls the abundance of monocytic MDSCs in the tumor microenvironment and how this relates to promotion of tumor metastasis. Our work establishes MIF as a potentially valuable therapeutic target in tumor metastasis and supports a potential alternative mechanism for chemoprevention in which SFN may target the inflammatory tumor microenvironment through inhibition of this cytokine.

Citation Format: Kendra D. Simpson, Dennis J. Templeton, Janet V. Cross. Macrophage Migration Inhibitory Factor (MIF) regulates monocytic MDSC abundance to contribute to tumor growth and metastasis. [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 2812. doi:10.1158/1538-7445.AM2013-2812
Abstract 2819: Role of endothelin 3 in melanoma progression and metastasis.
Authors: Chin Nikeisha L. , Gallegos Juan Carlos , Cruz Rosy , Garcia Ruslan , Gonzalez Andrea , Borobia Manuel , Kos Lidia
Published: August 2014

G-protein coupled receptors have been identified as important factors in melanoma progression including growth and proliferation of tumorigenic cells, as well as facilitating metastasis. Upregulation of the G-protein coupled receptor Endothelin receptor b (Ednrb) and its ligand Endothelin 3 (Edn3) has been implicated in melanoma progression in several ‘in vitro’ experiments. In this study we investigated the putative role of Edn3 over-expression in melanoma progression in vivo. Tg(Grm1) Epv mice over-expressing metabotropic glutamate receptor 1 (Grm1) under the Dopachrome tautomerase promoter that spontaneously produce melanocytic lesions in the ears and tails with limited metastatic capability, were crossed with transgenic mice that over-express Edn3 under the keratin 5 promoter (K5-Edn3). The Tg(Grm1)Epv/K5-Edn3 mice tumors appeared ∼ 3 months earlier and grew at a rate 3-4 times faster than the Tg(Grm1)/Epv control mice. Ninety-five percent of Dct-Grm1/ K5-Edn3 mice also displayed pigmented lesions in distant organs such as the lungs. Real Time PCR analysis showed higher expression levels of angiogenic related genes such as Hif-1α in the Dct-Grm1/K5-Edn3mice, when compared to controls. Our studies demonstrate that Edn3 over-expression enhances melanoma progression to a fully malignant state. Our preliminary data also suggests that Edn3 may be involved in tumor angiogenesis leading to faster melanoma tumor development.

Citation Format: Nikeisha L. Chin, Juan Carlos Gallegos, Rosy Cruz, Ruslan Garcia, Andrea Gonzalez, Manuel Borobia, Lidia Kos. Role of endothelin 3 in melanoma progression and metastasis. [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 2819. doi:10.1158/1538-7445.AM2013-2819

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 2828: miR-19a-3p inhibits breast carcinoma metastasis via reversing M2 phenotype of TAMs.
Authors: Yang Jian , Li Na , Zhang Zhuhong , Si Qin , Chen Chong , Liu Yan , Reisfeld Ralph A. , Sun Peiqing , Stupack Dwayne , Xiang Rong , Luo Yunping
Published: August 2014

Tumor-associated macrophages (TAMs), most of which exhibit M2 phenotype, function as immunosuppressive cells in tumor microenvironment (TME). Polarization of TAMs from a pro-immune (M1 like) phenotype to an immune-suppressive (M2-like) phenotype is one of the hallmarks of malignancy, but their molecular basis is still remains unknown. It has been reported that microRNAs are involved in monocyte-macrophage differentiation. In this study, we found that miR-19a-3p, broadly conserved in vertebrate, could reverse the M2 phenotype of RAW macrophage cells. When mouse breast tumor cells such as 4T1, 4TO7 and EMT6 were co-cultured with RAW macrophage cells which over express miR-19a-3p, the invasion capacity was suppressed. Meanwhile, when the conditional medium of RAW cells which were transfected with miR-19a-3p mimic was added into culturing medium of tumor cells, the migration capacity of 4T1 and EMT6 breast cancer cells was inhibited. Moreover, when miR-19a-3p was injected intratumor, consistent with the in vitro experiments, we found that the M2 phenotype of TAMs was suppressed significantly. Although 4T1 xengraft breast tumor growth was not affected by miR-19a-3p, lung metastasis of tumor cells was significantly suppressed. Taken together, our findings indicate that miR-19a-3p is down-regulated in M2 phenotype RAW macrophage and TAMs in TME. The low expression of miR-19a-3p plays an important role in inducing M2 macrophage polarization and promoting migration and invasion capacity and metastasis.

Citation Format: Jian Yang, Na Li, Zhuhong Zhang, Qin Si, Chong Chen, Yan Liu, Ralph A. Reisfeld, Peiqing Sun, Dwayne Stupack, Rong Xiang, Yunping Luo. miR-19a-3p inhibits breast carcinoma metastasis via reversing M2 phenotype of TAMs. [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 2828. doi:10.1158/1538-7445.AM2013-2828

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 2829: Patient-derived primary melanoma cells that differentially express melano-specific antigens as a model for vaccines.
Authors: Suriano Robert , George Andrea L. , Rajoria Shilpi , Geliebter Jan , Tiwari Raj K. , Wallack Marc
Published: August 2014

Transformation of pigment producing melanocytes into melanoma is a complex multi-step process involving the generation and/or over expression of various immunotherapeutic antigens. Several of these melanoma associated antigens (MAAs) are directly involved in melanin biosynthesis and melanosome biogenesis. The precise contribution of proteins involved in melanin synthesis to the MAA associated immune response is poorly understood. To this end, we have developed primary cell lines from human patients and xenograft models to analyze melanin synthesis and antigen expression. In vitro, from the perspective of melanin production, these primary cell lines express essential proteins associated with melanin synthesis, namely tyrosinase, Trp-1, Trp-2, and gp100, all of which are immunotherapeutic targets in melanoma. When grafted in nude mice, these cell lines were capable of producing solid pigmented tumors in vivo. However, three out of the five cell lines that produced melanin in vivo failed to produce melanin in vitro, even when cultured with supplementation of either α-melanocyte stimulating hormone (MSH), basic fibroblast growth factor (bFGF), or matrigel. Despite expression of tyrosinase, Trp-1, Trp-2, and gp100, two of the five cell lines failed to grow in nude mice or produce melanin in vitro. These patient derived primary cells and xenografts were also analyzed for expression of various other significant MAAs, which included MART-1 (Melan-A), MAGE-A1, and NY-ESO-1 as well as antigens not classified as MAAs but essential for tumor progression such as CD71 (transferring receptor) and CD146 (MCAM or melanoma cell adhesion molecule). All of the antigens analyzed in our cell lines, such as gp100, NY-ESO-1, MART-1, MAGE-A1, have been the targets of clinical trials but our results clearly indicate that these antigens are differentially expressed by our primary melanoma cells. Therefore, we believe that in order to develop a clinically effective immunotherapeutic vaccine, multiple cell lines expressing a combination of melano-specific antigens and a repertoire of MAAs that allows for minimal immune evasion may be necessary. To this end, we are exploring a vaccinia virus antigen retrieval technology, which incorporates multiple primary cell lines to develop such a pan-antigen melanoma vaccine.

Citation Format: Robert Suriano, Andrea L. George, Shilpi Rajoria, Jan Geliebter, Raj K. Tiwari, Marc Wallack. Patient-derived primary melanoma cells that differentially express melano-specific antigens as a model for vaccines. [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 2829. doi:10.1158/1538-7445.AM2013-2829
Abstract 2831: Induction of CXCL9/Mig expression in the tumor microenvironment promotes protective antitumor immune responses.
Authors: Gorbachev Anton , Kish Danielle , Fairchild Robert
Published: August 2014

Despite intense efforts to design immunotherapeutic strategies, the success of cancer immunotherapy remains extremely limited. Major factors limiting immune responses to tumors include insufficient activation of tumor-reactive effector T cells, poor intra-tumor recruitment of effector T cells, and/or suppression of their functions within the tumor. One mediator involved in effector T cell activation and recruitment to inflammatory sites is the T cell chemoattractant chemokine CXCL9/Mig. Our recent studies in murine models of aggressive skin cancer (cutaneous sarcoma and melanoma) have indicated that these tumors cease the production of CXCL9/Mig as a result of immunoediting mediated by T cells and IFN-g. The consequence of Mig deficiency in the tumor microenvironment is the increased resistance of immunoedited Mig-deficient tumor variants to T cell-mediated immunity. Here we demonstrated that inducing constitutive expression of Mig within Mig-deficient tumors by delivery of Mig-encoding retroviral vector promotes protective anti-tumor immune responses and results in rejection of cutaneous tumors. This modulation of intra-tumor Mig expression induced potent tumor-specific CD4 and CD8 T cell-mediated responses capable of complete tumor elimination and subsequent protection to tumor re-challenge. Furthermore, the delivery of Mig-overexpressing tumor cells as a vaccine into mice with established cutaneous tumors induced systemic anti-tumor immune responses that suppressed growth of established tumors. This therapeutic effect correlated with the presence of dendritic cells (DC) in the tumor-draining lymph nodes (TDLN) that were very potent in the activation of tumor antigen-specific IFN-g producing T cells. These results suggest that besides the recruitment of effector T cells into the tumor, tumor-derived Mig enhances activation of these T cells possibly by affecting tumor-infiltrating DC that acquire tumor antigens and then migrate into TDLN to prime tumor-specific effector T cells. Therefore, Mig might be a potent component of vaccines aimed to boost anti-tumor immunity in patients with aggressive skin cancers deficient in the expression of this chemokine.

Citation Format: Anton Gorbachev, Danielle Kish, Robert Fairchild. Induction of CXCL9/Mig expression in the tumor microenvironment promotes protective antitumor immune responses. [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 2831. doi:10.1158/1538-7445.AM2013-2831
Abstract 2839: NK cell response and tumor debulking are associated to prolonged survival in recurrent glioblastoma treated by dendritic cells loaded with autologous tumor lysate.
Authors: Pellegatta Serena , Eoli Marica , Frigerio Simona , Antozzi Carlo , Bruzzone Maria Grazia , Cantini Gabriele , Nava Sara , Anghileri Elena , Cuppini Lucia , Cuccarini Valeria , Ciusani Emilio , Dossena Marta , Pollo Bianca , Mantegazza Renato , Parati Eugenio A. , Finocchiaro Gaetano
Published: August 2014

Recurrent glioblastoma (GB) are highly aggressive tumors allowing 6-8 month survival. Here we evaluated the possible benefits of immunotherapy with mature dendritic cells (DC) loaded with autologous tumor lysate in 15 patients with recurrent GB. After a median follow-up of 8 months median progression free survival (PFS) was 4.4 months and median overall survival (OS) 8.0 months. Patients with small tumors at first vaccination (< 20 cc; n = 8) had significantly longer PFS and OS than others: 6.0 vs 3.0 months (p = 0.01) and 16.5 vs 7.0 months (p = 0.003), respectively. Patients were analysed for CD8+ T cells, CD56+ NK cells and other relevant immunological parameters in peripheral blood before and after immunization, defining a vaccination/baseline ratio (V/B ratio). Increased V/B ratio of NK but not CD8+ T cells was significantly associated to longer PFS and OS. Patients showing NK cell responses had higher levels of IFN-γ and E4BP4, the NK cell transcription factor. Furthermore, NK V/B ratio was inversely correlated with TGF-β2 and VEGF V/B ratio. The results suggest that tumor-loaded DC may increase survival of recurrent GB after effective tumor debulking and emphasize the role of NK cell responses in this therapeutic setting.

Citation Format: Serena Pellegatta, Marica Eoli, Simona Frigerio, Carlo Antozzi, Maria Grazia Bruzzone, Gabriele Cantini, Sara Nava, Elena Anghileri, Lucia Cuppini, Valeria Cuccarini, Emilio Ciusani, Marta Dossena, Bianca Pollo, Renato Mantegazza, Eugenio A. Parati, Gaetano Finocchiaro. NK cell response and tumor debulking are associated to prolonged survival in recurrent glioblastoma treated by dendritic cells loaded with autologous tumor lysate. [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 2839. doi:10.1158/1538-7445.AM2013-2839

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 284: Down-regulated Kruppel-like factor 4 induces the migration and epithelial-mesenchymal transition in human hepatoma cells.
Authors: Sung Ming-Ta , Lee Hsin-Chen , Hsia Cheng-Yuan , Chi Chin-Wen
Published: August 2014

The transcriptional factor Kruppel-like factor 4 (KLF4) plays important roles in development, stemness, and tumorigenesis. Several studies have suggested that KLF4 is important in various tumors; however limited information is available on the detailed function of KLF4 in hepatocellular carcinoma. The objective of this study was to examine the functional roles of KLF4 in metastasis of hepatoma cells. In order to investigate the role of KLF4 in the progression of hepatocellular carcinoma, we used lentiviral transduction method to down-regulate KLF4 expression in Hep3B and HA22T/VGH hepatoma cell lines. The effects of KLF4 on cell migration, invasion and the expression of epithelial-mesenchymal transition (EMT) related proteins were evaluated in KLF4-knockdown hepatoma cells. Transwell migration assay and matrigel invasion assay revealed the migration and invasion abilities were significantly increased in KLF4-knockdown Hep3B or HA22T/VGH cells. Knockdown KLF4 in hepatoma cells resulted in decreased mRNA and protein expression of E-cadherin, and a concomitant increase of vimentin, MMP2 and MMP9. These results corresponded well with our finding that overexpression of KLF4 in Mahlavu hepatoma cells led to significant inhibition of cell migration and invasion through up-regulating E-cadherin and down-regulating vimentin, MMP2 and MMP9. Snail and Bmi1, repressors of E-cadherin gene expression, were also markedly decreased in KLF4-overexpressing Mahlavu cells. Therefore, these results together suggested that KLF4 is tightly involved in the epithelial-mesenchymal transition and metastasis in the tumor progression of hepatocellular carcinoma.

Citation Format: Ming-Ta Sung, Hsin-Chen Lee, Cheng-Yuan Hsia, Chin-Wen Chi. Down-regulated Kruppel-like factor 4 induces the migration and epithelial-mesenchymal transition in human hepatoma cells. [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 284. doi:10.1158/1538-7445.AM2013-284
Abstract 286: Cucurbitacin B inhibits epithelial-to-mesenchymal transition in breast cancer cells through Snail downregulation.
Authors: Gupta Parul , Srivastava Sanjay K.
Published: August 2014

Metastasis is a primary cause of about 90% of cancer related deaths worldwide. It is associated with epithelial to mesenchymal characteristics transition (EMT), which helps in attaining migratory phenotype by tumor cells. Cucurbitacin B (CuB), a triterpenoid steroidal compound has been shown to suppress JAK/STAT3 signaling and cause G2/M arrest in cancer cells. However, the role of CuB as an anti-metastatic agent still remains to be elucidated. In the present study, we investigated the effects of CuB on EMT in breast cancer cells. These cells are known to exhibit significant mesenchymal properties that endow aggressive nature. Our results show that CuB significantly suppressed cell migration of MDA-MB-231 cells in wound healing assay. In addition, we also observed reduced cell invasion with CuB treatment using transwell invasion assay. To elucidate the mechanism of these effects, western blotting was performed using whole cell lysates from CuB treated MDA-MB-231 cells. We observed significantly reduced expression of snail and twist with CuB treatment. Snail and twist are known to be repressors of E-cadherin. Generally upregulation of snail and twist mark the initiation of EMT, and eventually leads to loss of epithelial signature of cells. Furthermore, with CuB treatment upregulation of E-cadherin was observed. Transforming growth factor β is known to induce EMT in malignant breast cancer cells. We further investigated the role of CuB on TGFβ induced EMT. Interestingly, we observed that TGFβ induced snail expression was reversed by CuB in MDA-MB-231 cells. Moreover, CuB treatment also contributed protection to the loss of E-cadherin caused by TGFβ treatment. Taken together our results indicate reversal of EMT by CuB treatment in breast cancer cells, which lead to suppression of metastasic ability. However, further in vitro and in vivo mechanistic studies are under progress. [Supported in part by R01 grant CA129038 (to S.K.S) awarded by the National Cancer Institute].

Citation Format: Parul Gupta, Sanjay K. Srivastava. Cucurbitacin B inhibits epithelial-to-mesenchymal transition in breast cancer cells through Snail downregulation. [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 286. doi:10.1158/1538-7445.AM2013-286
Abstract 2861: A novel negative regulation principle between DAPK and STAT3 in inflammation-associated carcinogenesis.
Authors: Chakilam Saritha , Gandesiri Muktheswar , Rau Tilman , Neufert Clemens , Agaimy Abbas , Mahadevan Vijayalakshmi , Ivanovska Jelena , Wirtz Ralph , Benderska Natalya , Schulze-Lührmann Jan , Wittkopf Nadine , Chellappan Ajithavalli , Ruemmele Petra , Vieth Michael , Rave-Fränk Margret , Christiansen Hans , Hartmann Arndt , Atreya Raja , Becker Christoph , Steinberg Pablo , Schneider-Stock Regine
Published: August 2014

It has been shown that the TNF-IL-6-STAT3 pathway plays a crucial role in promoting ulcerative colitis associated carcinoma (UCC). As inflammation compromises gut homeostasis and is associated with cancer progression, it is important to understand the role of key signalling molecules that are involved in the activation of the inflammatory cascade. Interestingly, we found that intestinal epithelial cells (IEC) of UCC in comparison to ulcerative colitis (UC) show high expression level of the anti-inflammatory Death associated protein kinase (DAPK) but only low levels of pSTAT3. Accordingly, epithelial DAPK expression was enhanced in STAT3IEC-KO mice. To unravel a possible regulatory mechanism we used an in vitro model of TNF-treated IEC. We identified a new function of DAPK in suppressing TNF-induced STAT3 activation as DAPK si knockdown and treatment with a DAPK inhibitor potentiated STAT3 activation and IL-6 secretion. DAPK attenuated STAT3 activity directly by physical interaction shown in a 3D structural model. This model suggests that DAPK induced conformational changes in the STAT3 dimer masked its nuclear localization signal. Vice versa, pharmacologic inactivation of STAT3 led to an increase in DAPK mRNA and protein levels. Chromatin immunoprecipitation data showed that STAT3 restricted DAPK expression by promoter binding, thereby reinforcing its own activation by inducing IL-6. This novel negative regulation principle was confirmed in an AOM-DSS-colon carcinogenesis mouse model. It might balance TNF-induced inflammation and seems to play an important role in the inflammation-associated transformation process. DAPK as a negative regulator of STAT3 emerges as therapeutic option in the treatment of UC and UCC.

Citation Format: Saritha Chakilam, Muktheswar Gandesiri, Tilman Rau, Clemens Neufert, Abbas Agaimy, Vijayalakshmi Mahadevan, Jelena Ivanovska, Ralph Wirtz, Natalya Benderska, Jan Schulze-Lührmann, Nadine Wittkopf, Ajithavalli Chellappan, Petra Ruemmele, Michael Vieth, Margret Rave-Fränk, Hans Christiansen, Arndt Hartmann, Raja Atreya, Christoph Becker, Pablo Steinberg, Regine Schneider-Stock. A novel negative regulation principle between DAPK and STAT3 in inflammation-associated carcinogenesis. [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 2861. doi:10.1158/1538-7445.AM2013-2861

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 2864: Molecular imaging of early inflammatory response during hyperplasia and/or colitis in response to bacterial infection.
Authors: Papineni Rao , Ahmed Ishfaq , LeVine Steven , Bradley Kenneth , Mclaughlin William , Pizzonia John , Umar Shahid
Published: August 2014

Inflammatory diseases such as ulcerative colitis recruit myeloid cells and result in inflammatory responses such as generation of reactive oxygen species (ROS). Development of non-invasive luminescence imaging approaches is needed to evaluate these inflammatory responses. We developed methodologies to show that vibration induced gut inflammation can be determined using luminescence imaging approach in a non-invasive fashion (Papineni and Vizard, World Molecular Imaging Congress

2012). Citrobacter rodentium (CR) is a natural bacterial pathogen that infects the distal colon of mice and induces transmissible murine colonic hyperplasia (TMCH). Here we determined the early inflammatory response (ROS activity) to intestinal infection by CR in a longitudinal study. We further assessed the effects of allelic variation on murine chromosome 11 (B6.CAST. 11) on the inflammatory response. The ROS activity was monitored in real-time in vivo using L-012 (8-amino-5-chloro-7-phenylpyridol [3,4-d]pyridazine-1,4(2H,3H) dione), a chemiluminescence reporter, using planar multimodal imaging system. 0.1 ml of 1 mg/ml L-012 probe was injected (i.p.) in control and the experimental mice that were subjected to CR infection. Necrosis was imaged (post-infection day-19) using a fluorescent probe containing a bis (zinc2+dipicolylamine, Zn-

DPA) motif which binds with high affinity to apoptotic, necrotic, and bacterial surfaces. Significant difference in the basal gut ROS activity was found in mice with allelic variation on chromosome 11 compared to the wild type littermates. Robust elevation in ROS activity was observed in both the wild type and B6.CAST.11mice that were infected with CR (post infection 9, 12, 19 days). The variation in the levels of ROS activity however was very distinct between the wild type and B6.CAST.11 mice indicating the contribution of molecular determinants at the chromosome 11. The potential advantages in these real-time ROS monitoring methodologies by in vivo imaging, in understanding mechanisms of infection, inflammation, and development of better intervention strategies will be elaborated.

Citation Format: Rao Papineni, Ishfaq Ahmed, Steven LeVine, Kenneth Bradley, William Mclaughlin, John Pizzonia, Shahid Umar. Molecular imaging of early inflammatory response during hyperplasia and/or colitis in response to bacterial infection. [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 2864. doi:10.1158/1538-7445.AM2013-2864