bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3916: Differential roles of microglia and macrophages for glioma progression.
Authors: Osswald Matthias , Gramlich Miriam , Liao Yunxiang , Wick Wolfgang , Winkler Frank
Published: August 2014

Glioblastomas belong to the tumors with the worst prognosis. One reason for the short survival time after diagnosis is the invasiveness of these tumors, which makes a total resection impossible, and hinders effective local therapies. So far, the exact role of the resident microglia and blood macrophages in the process of tumor cell invasion remains unclear. We investigated the role of these immune cells using CX3CR1 transgenic BL/6 mice. In heterozygous CX3CR1-GFP mice, the function of microglia or macrophages is not impaired but both cell types can be tracked over time by stable GFP expression; homozygous mice have both GFP-labeled microglia and macrophages, but lack a functional CX3CR1 chemokine receptor. Both mice strains received a chronic cranial window with subsequent stereotactic Gl261 glioma cell injection into the brain three weeks later. The invasion dynamics of the stably dsRed-transduced tumor cells was monitored using intravital two photon microscopy. In homozygous animals, tumor cells migrated faster than in heterozygous mice (4,6 μm/h vs. 3 μm/h, p<0.001). Both groups showed a perivascular tumor cell invasion pattern. Moreover, single microglia or macrophage cells showed intracellular RFP-positive fragments as a sign of glioma cell phagocytosis; this was observed only in heterozygous animals and not in homozygous animals lacking a functional CX3CR1 receptor. To decipher the role of resident microglia vs. macrophages in glioma invasion, blood macrophages were depleted in heterozygous CX3CR1 mice by intravenous injection of clodronate liposomes. In those mice tumor cell invasion was dramatically reduced. Pre-existing brain resident microglia was not affected by clodronate treatment. All in all, these results implicate a CX3CR1-dependent proinvasive role for blood-borne macrophages in glioma cell invasion in the live mouse brain, whereas residential microglia seems to exert anti-glioma effects. This microglia - macrophage dichotomy has implications for therapies aiming at inhibition of glioma invasion, via CX3CR1 targeting and beyond.

Citation Format: Matthias Osswald, Miriam Gramlich, Yunxiang Liao, Wolfgang Wick, Frank Winkler. Differential roles of microglia and macrophages for glioma progression. [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 3916. doi:10.1158/1538-7445.AM2013-3916
Abstract 3917: Utilizing optical imaging technologies to screen candidate ADC antibodies.
Authors: Lin Xin , Peterson Norman , Wilson George , Kamal Adeela , Harper Jay
Published: August 2014

Antibody-drug conjugates (ADCs) represent a promising new therapeutic option to treat cancer where powerful cytotoxic compounds are conjugated to antibodies targeting a tumor-associated antigen. Molecular imaging allows relatively quick, non-invasive assessment of biological and biochemical processes in living subjects and when applied to research and development of ADC therapeutics, it has the potential to expedite the drug development process. The current study aims to evaluate optical imaging as a tool to aid in the selection of antibody candidates by using imaging data to predict in vivo efficacy of ADC therapeutics. In this study, candidate antibodies with engineered cysteines for site-specific conjugation of payloads were successfully labeled with the near-infrared fluorescent dye, alexa fluor 750, to achieve a degree of labeling of 2 dyes per antibody. Mice bearing target-positive tumors were treated with the dye-labeled antibodies, and were imaged at 1, 2, and 6 days post-injection using a fluorescence molecular tomography (FMT) in vivo imaging system. The tumor and major organs were subjected to ex vivo optical imaging and confirmatory immunofluorescence staining. Differences were observed in the tumor uptake, biodistribution profiles, and penetration of the four candidate antibodies. Concurrently, in vivo efficacy studies are being conducted with ADCs comprised of these lead antibodies, and the imaging data, along with the binding affinity and internalization rate of each antibody, will be compared to the pharmacology data to determine if a predictive correlation exists. Once established, correlations between the imaging data and efficacy results would justify the use of molecular imaging to screen large panels of antibodies to aid in the lead selection for ADCs, thus enabling significant time and cost reduction for the development of ADC therapeutics.

Citation Format: Xin Lin, Norman Peterson, George Wilson, Adeela Kamal, Jay Harper. Utilizing optical imaging technologies to screen candidate ADC antibodies. [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 3917. doi:10.1158/1538-7445.AM2013-3917
Abstract 3922: A comparative study of affibody, antibody and EGF for near-infrared fluorescence imaging of EGFR- and EGFRvIII-expressing tumors.
Authors: Gong Haibiao , Kovar Joy L. , Cheung Lael , Rosenthal Eben L. , Olive David M.
Published: August 2014

Aberrant overexpression and/or activation of epidermal growth factor receptor (EGFR) is associated with many types of cancers. EGFR variant III (EGFRvIII) is a common in-frame deletion mutant, which lacks a large part of the extracellular portion (exons 2-7), including components of the ligand-binding domain. Although EGFR has been extensively studied as

a molecular imaging target, information about EGFRvIII-targeted molecular imaging is lacking. In this study, an EGFR-specific affibody, a therapeutic antibody (panitumumab) and the ligand EGF were labeled with IRDye 800CW (Ex/Em: 774/789 nm), and named as Aff800, Pan800 and EGF800 respectively. The binding affinities of the labeled agents were compared in cell-based assays using a rat glioma cell line F98 parental (F98-p) lacking EGFR, and two F98-derived transgenic cell lines expressing EGFR or EGFRvIII (designated as F98-EGFR and F98-vIII respectively). Results showed that all agents could bind to F98-EGFR, with Pan800 having the highest binding affinity, followed by Aff800 and EGF800. Pan800 and Aff800, but not EGF800, bound to F98-vIII with similar affinities. In vivo animal imaging studies demonstrated that compared to F98-p tumor signals, F98-EGFR tumor generated higher signals for all three agents. However, in the case of F98-vIII, only Pan800 and Aff800 signals were higher. Analysis of tissue lysates showed that a large portion of Pan800 was degraded into small fragments in F98-EGFR and F98-vIII tumors, possibly due to proteolytic digestion after its specific binding and internalization. In conclusion, Pan800 and Aff800 could be used as imaging agents for both wild type EGFR and EGFRvIII, whereas EGF800 only targets wild type EGFR.

Citation Format: Haibiao Gong, Joy L. Kovar, Lael Cheung, Eben L. Rosenthal, David M. Olive. A comparative study of affibody, antibody and EGF for near-infrared fluorescence imaging of EGFR- and EGFRvIII-expressing tumors. [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 3922. doi:10.1158/1538-7445.AM2013-3922
Abstract 3940: Ezrin is a key regulator in Src-induced angiogenesis and lymphangiogenesis in breast cancer.
Authors: Ghaffari Abdi , Hoskin Victoria , Szeto Alvin , Hum Maaike , Liaghati Navid , Nakatsu Kanji , Elliott Bruce E.
Published: August 2014

Src kinase plays a critical intrinsic role in metastasis through disruption of cell-cell junctions and focal adhesions, and extrinsically by upregulation of paracrine pro-angiogenic factors such as VEGF. Ezrin, a Src substrate and membrane cytoskeletal crosslinker, acts cooperatively with Src in the regulation of malignant phenotypes in carcinoma cells. Ezrin expression has been associated with increased metastatic potential in various human cancers and it has also been identified as a potential prognostic/predictive biomarker for an invasive subtype of breast cancer. To examine the extrinsic role of ezrin in Src-induced tumour vascularization we performed Matrigel plug assays by injecting human mammary carcinoma cells subcutaneously into the flanks of immunodeficient Rag2-/-γc-/- and nude mice. On day 12 post-injection, intravital imaging revealed a potent angiogenic response in MDA-MB-231 cells expressing activated Src (MDASrc). However, ezrin knockdown (KD) in MDASrc cells was sufficient to reverse Src-induced tumour vascularization. Microvascular density, vascular cross-sectional area, and blood (CD31) and lymphatic (Lyve-1 & podoplanin) vessel immunostaining of harvested Matrigel plugs (day 12) further confirmed the intravital results. In addition, ectopic expression of a mutant ezrin (Y477F), which is non-phosphorylatable by Src, also caused significant reduction in tumour neovascularization. We further assessed the angiogenic activity of conditioned medium collected from various cell lines by performing ex vivo aortic ring assays. Again, Src-induced endothelial cells branching in MDASrc group was significantly reduced following ezrin KD. To assess lymphangiogenesis, we co-cultured primary lymphatic endothelial cells (hLEC) with MDASrc in presence and absence of ezrin. Ezrin KD significantly reduced Src-induced hLEC tube formation, migration, and permeability in vitro. Results from conditioned media and co-culture assays also suggested the involvement of paracrine angio- and lymphangio-genic factors regulated by Src/ezrin signaling axis in cancer cells. Western blot analysis of cell lysates revealed that ezrin KD was sufficient to block Src-induced Stat3 activation and VEGF-A and -C expression in MDASrc cells. In conclusion, our study provides a novel mechanism for the role of ezrin in breast cancer metastasis as a key regulator in Src-induced angio- and lymphangio-genesis, and could lead to improved drug targeting of metastatic disease.

Citation Format: Abdi Ghaffari, Victoria Hoskin, Alvin Szeto, Maaike Hum, Navid Liaghati, Kanji Nakatsu, Bruce E. Elliott. Ezrin is a key regulator in Src-induced angiogenesis and lymphangiogenesis in breast cancer. [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 3940. doi:10.1158/1538-7445.AM2013-3940
Abstract 3945: IKK-Epsilon, a novel factor identified by proteomics, contributes to prostate cancer cell-induced osteoclast activity in vitro.
Authors: Yang Xin , Xiao Zhen , Wang Jianhua , Lu Yi , Xu Yang , Huang Xin , Mizokami Atsushi , Keller Evan T. , Zhang Jian
Published: August 2014

Prostate cancer preferentially metastasizes to skeleton resulting in osteoblastic lesions with underlying osteoclast activity. Novel strategies to inhibit the tumor skeletal metastasis involve targeting not only tumor cells but also bone microenvironment which is composed of osteoclasts, osteoblasts and stromal cells that include fibroblasts, immune and inflammatory cells, adipocytes, glial cells, smooth muscle cells, and resident and recruited vascular cells. The microenvironment can exert both stimulatory and inhibitory influences on proliferation and malignant properties of tumor cells. In this study, we intended to identify factors that may play key roles in this bidirectional and dynamic interaction between tumor cells and bone microenvironment. First, we fractionated the prostate cancer PC3 cell conditioned media by the molecular weight of proteins in media using centricon column. Then we applied the fractions to culturing osteoclast precursor RAW 264.7 cells in vitro. We repeated and scaled up the above steps to collect enough conditioned media, and concentrated the fractions of interest. The fractions that showed activity of osteoclast induction were determined by mass spectrometry. We found that the conditioned media contained a large amount of IKK-Epsilon, a subunit of PMA-inducible IKK (I-KappaB Kinase) complex. We further observed that recombinant IKK-Epsilon could induce osteoclast activity in vitro, but unexpectedly it had no effect on the activation of NF-kappa-B by TNF or IL-1 in these cells. Together with the findings that several identified factors in the conditioned media such as MCP-1 and IL-8 that induced osteoclast activity, in vitro validation of the identified factor IKK-Epsilon was confirmed by using neutralizing antibody on the functional assays. We have not yet identified unknown factors in the current study, however it is clear that unknown factors in the conditioned media may also play key roles in the tumor-induced osteoclast activation since blocking MCP-1, IL-8, and IKK-Epsilon did not completely block the tumor-induced osteoclast activity. We conclude that IKK-Epsilon, a novel factor identified by proteomics, may contribute to prostate cancer cell-induced osteoclast activity in vitro. Supported by NSF key project 81130046; NSF projects 81171993 and 81272415; Guangxi projects 201201ZD004 and 2012GXNSFCB53004.

Citation Format: Xin Yang, Zhen Xiao, Jianhua Wang, Yi Lu, Yang Xu, Xin Huang, Atsushi Mizokami, Evan T. Keller, Jian Zhang. IKK-Epsilon, a novel factor identified by proteomics, contributes to prostate cancer cell-induced osteoclast activity in vitro. [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 3945. doi:10.1158/1538-7445.AM2013-3945
Abstract 3947: AMG 161, a fully human monoclonal antibody to human RANKL, inhibits tumor-induced osteoclastogenesis and reduces skeletal tumor burden in mice that express chimeric (murine / human) RANKL.
Authors: Canon Jude R. , Bryant Rebecca , Roudier Martine , Dougall William C.
Published: August 2014

Tumor cells can induce RANKL expression in the bone stroma, causing activation of osteoclasts which consequently mediate bone breakdown and subsequent tumor proliferation. RANKL is an essential mediator of osteoclast function and survival. RANKL inhibition by OPG-Fc can prevent tumor-induced osteolysis, decrease skeletal tumor burden, and increase survival in preclinical models. Denosumab is an anti-huRANKL antibody approved for the prevention of skeletal-related events due to bone metastases from solid tumors. Selective anti-huRANKL antibodies do not bind murine RANKL, therefore efficacy cannot be tested in typical mouse models. We developed a breast cancer bone metastasis model in mice in which exon 5 from murine RANKL was replaced with its human ortholog (“mu/huRANKL”). We tested the effect of AMG 161, an antibody which binds huRANKL and chimeric mu/huRANKL, on tumor burden and associated osteoclasts. After intra-cardiac injection of MCF-7Luc breast carcinoma cells, hind limb metastases formed and tumor burden was tracked by bioluminescence imaging (BLI). AMG 161 (0.3 or 3.0 mg/kg, 2x/week), OPG-Fc (3.0 mg/kg, 2x/week) or vehicle were administered beginning 10 days post tumor injection to treat established bone metastases until study termination at day 31. AMG 161 resulted in a dose-dependent decrease in skeletal tumor burden (3.0 mg/kg: 84% decrease vs. control p=0.0052) similar to that observed with OPG-Fc (85% decrease vs. control, p=0.0038). Histological analysis at the end of the study showed reductions in skeletal tumor burden consistent with the BLI. AMG 161 treatment resulted in a significant decrease in tumor-associated osteoclasts (TRAP staining) and inhibition of osteolytic lesions (X-ray). The inhibition of tumor burden by the anti-RANKL antibody was indirect via osteoclast inhibition since MCF-7Luc cells do not express RANK, and RANKL inhibition does not affect subcutaneous growth of MCF-7Luc cells. These data support the efficacy of anti-RANKL antibody treatment to inhibit tumor-induced osteoclastogenesis and to block the growth of bone metastases.

Citation Format: Jude R. Canon, Rebecca Bryant, Martine Roudier, William C. Dougall. AMG 161, a fully human monoclonal antibody to human RANKL, inhibits tumor-induced osteoclastogenesis and reduces skeletal tumor burden in mice that express chimeric (murine / human) RANKL. [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 3947. doi:10.1158/1538-7445.AM2013-3947
Abstract 395: Farnesyl diphosphate farnesyltransferase induces metastasis via upregulating secreted phosphoprotein 1 in non-small cell lung cancer.
Authors: Yang Yi-Fang , Jan Yi-Hua , Hsiao Michael
Published: August 2014

Identifying cancer progression associated genes that predicting patient outcome might provide opportunities for developing better therapeutic strategies for lung cancer patients. In the present study, we investigated whether farnesyl diphosphate farnesyltransferase (FDFT1), an enzyme of mevalonate pathway, is a potential biomarker of lung cancer metastasis. We further investigated its prognostic value and functional role in lung cancer invasion and metastasis. Our results by immunohistochemistry (IHC) analysis showed the expression of FDFT1 was significantly higher in tumors compared to normal lung tissues. Overexpression of FDFT1 was significantly associated with tumor size (P < 0.001), advanced tumor stage (P <0.001), lymph node metastasis (P = 0.02), distal metastasis (P = 0.043), and poor prognosis (P <0.001). Loss of FDFT1 expression significantly suppressed invasion and migration potential of lung cancer cell lines, whereas FDFT1 overexpression promoted invasion and migration in vitro and in vivo. Microarray analysis further identified secreted phosphoprotein 1 (SPP1) as an important mediator for FDFT1-induced invasion and migration. Furthermore, patients with high FDFT1 and high SPP1 expression showed dismal outcomes as compared to patients with low FDFT1 and low SPP1 expression with good prognosis. In conclusion, we identified the FDFT1 in this study as a new lung cancer progression biomarker that is associated poor prognosis and its overexpression resulted in enhanced tumor invasion and metastasis through upregulation of SPP1. Our findings may implicate the possibility of targeting FDFT1 as a novel therapeutic strategy to inhibit lung cancer metastasis.

Citation Format: Yi-Fang Yang, Yi-Hua Jan, Michael Hsiao. Farnesyl diphosphate farnesyltransferase induces metastasis via upregulating secreted phosphoprotein 1 in non-small cell lung cancer. [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 395. doi:10.1158/1538-7445.AM2013-395

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 3951: Imaging exosome transfer from colon cancer cells to stroma at liver metastatic sites in nude mouse.
Authors: Suetsugu Atsushi , Homma Kimi , Saji Shigetoyo , Moriwaki Hisataka , Ochiya Takahiro , Hoffman Robert M.
Published: August 2014

Exosomes play an important role in cell-to-cell communication to promote tumor metastasis. In order to image the fate of cancer-cell-derived exosomes in liver metastasis of colon cancer, we used green fluorescent protein (GFP)-tagged CD63, which is a general marker of exosomes. GFP-exosome producing RFP human colon cancer HCT cells (HCT-RFP/GFP- Exo cells) were injected in the spleen of nude mice. By day 28, HCT-RFP/GFP-Exo cells were visualized in the liver with the Olympus OV100 imaging system. HCT-RFP/GFP- Exo cells secrete GFP-exosomes in the liver metastasis site as observed with the Olympus FV1000 confocal microscope. Colon cancer cells secreted exosomes into the tumor microenvironment. Tumor-derived exosomes were incorporated into tumor-associated cells as well as circulating in the blood of mice with colon cancer metastases. These results suggest that tumor-derived exosomes may contribute to forming a niche to promote tumor growth and metastasis. Our results demonstrate the usefulness of GFP imaging to investigate the role of exosomes in colon cancer liver metastasis.

Citation Format: Atsushi Suetsugu, Kimi Homma, Shigetoyo Saji, Hisataka Moriwaki, Takahiro Ochiya, Robert M. Hoffman. Imaging exosome transfer from colon cancer cells to stroma at liver metastatic sites in nude mouse. [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 3951. doi:10.1158/1538-7445.AM2013-3951
Abstract 3955: The tumor suppressor C/EBPdelta promotes hypoxia-induced EMT and expression of stem cell markers through inhibition of FBXW7: Implications for mammary tumor metastasis.
Authors: Kuppusamy Balamurugan , Sterneck Esta
Published: August 2014

The transcription factor CCAAT/enhancer binding protein delta (C/EBPδ) exhibits tumor-suppressor like-qualities such as downregulation of cyclin D1, and promotion of growth arrest and apoptosis. However, using a mouse model of mammary tumorigenesis (MMTV-Neu), we had found that C/EBPδ has dual functions. C/EBPδ suppresses primary tumor incidence while also augmenting metastatic progression. The pro-metastatic function of C/EBPδ can be correlated with a role in hypoxia adaptation and expression of pro-inflammatory genes and macrophage activation. C/EBPδ exerts both of these functions through inhibition of FBXW7, a component of SCF E3 ubiquitin ligase complex, which targets substrates such as c-myc and Notch for degradation (Balamurugan et al, 2010; Balamurugan et al., under revision). Hypoxia is believed to promote tumor progression in part by inducing epithelial-mesenchymal transition (EMT), which contributes to the generation of stem-like cancer cells (CSCs). In the present study, we found that hypoxia-induced EMT and expression of CSCs markers were compromised in Cebpd null primary tumor cells and in human breast tumor cells when C/EBPδ expression was silenced. On the other hand, silencing of the C/EBPδ-repressed gene FBXW7 was sufficient to induce a mesenchymal-like phenotype. Loss of FBXW7 has been shown to promote acquisition of stemness. In line with this notion, we found that overexpression of FBXW7 in the breast tumor cell line MDA-MB-468 reduced expression of aldehyde dehydrogenase 1 (Aldh1), C/EBPδ and the active form of NOTCH (NICD). Together, these data suggest that C/EBPδ augments - whereas its target FBXW7 prevents - EMT and breast cancer stem cell development, which may be an additional mechanism by which C/EBPδ promotes metastatic progression. Our current investigations are aimed at understanding how the different functions of C/EBPδ are integrated depending on cellular environment and context that results in its tumor promoting function despite its tumor suppressor role.

Citation Format: Balamurugan Kuppusamy, Esta Sterneck. The tumor suppressor C/EBPdelta promotes hypoxia-induced EMT and expression of stem cell markers through inhibition of FBXW7: Implications for mammary tumor 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 3955. doi:10.1158/1538-7445.AM2013-3955
Abstract 3959: MMP13 contributes to metastatic tumor growth in the steatotic liver microenvironment.
Authors: Mendonsa Alisha M. , VanSaun Michael , Gorden David L.
Published: August 2014

The expanding obesity epidemic has led to an escalation in the incidence of non-alcoholic fatty liver disease (NAFLD) and steatohepatitis, which are associated with an increase in incidence of primary and metastatic liver cancer. As NAFLD progresses, it is characterized by marked inflammation in the liver described as non-alcoholic steatohepatitis (NASH). The mechanisms by which NASH increases risk of liver cancer are not fully known but complete understanding of these mediators is necessary for effective therapeutic and preventive strategies. Importantly, metastasis represents the final stage in cancer progression and is the most significant cause of cancer related mortality. Using our mouse model of diet induced steatosis, coupled with a model of experimental liver metastasis, we have previously shown a significant increase in the number of metastatic foci in the steatotic liver compared to the normal livers. Microarray analysis revealed that MMP13, a member of the Matrix Metalloproteinase (MMP) family recognized to be involved in liver disease and cancer progression, is significantly upregulated in the steatotic liver compared to normal livers. Additionally, MMP13 is expressed in several cancer cell types. Emerging data suggests that MMP13 may play a key role in priming the microenvironment for the establishment of liver metastases, by modulation of cytokine profiles and recruitment of inflammatory cells. . We hypothesize that MMP13 is a key modulator of the steatotic microenvironment, and that increased MMP13 levels favor primary tumor growth as well as the establishment of metastases. To determine the role of stromal MMP13 on metastasis to the liver, we are using the experimental splenic injection model of liver metastasis with syngeneic MC38 colon cancer cells in mice genetically deficient in MMP13. Additionally, since the tumor cells themselves express MMP13, wildtype mice are injected with either shRNA control or MMP13 knockdown MC38 colon cancer cells to determine the role of tumor cell derived MMP13. Preliminary data shows localization of MMP13 expression to the hepatic stellate cells as well as CD11b positive myeloid cells from livers with NASH. Further, invitro analysis of migration and invasion ability of parental and MMP13 shRNA knockdown MC38 cells show that MMP13 promotes the invasive and migratory properties of the metastatic cancer cells themselves. MMP13 may thus be a potential mediator of inflammation in the tumor microenvironment as well as of the metastatic capacity of cancer cells to the liver.

Citation Format: Alisha M. Mendonsa, Michael VanSaun, David L. Gorden. MMP13 contributes to metastatic tumor growth in the steatotic liver microenvironment. [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 3959. doi:10.1158/1538-7445.AM2013-3959
Abstract 396: Osteopontin-c splicing isoform contributes to ovarian cancer progression.
Authors: Tilli Tatiana M. , Mello Kivvi D. , Franco Vanessa , Robbs Bruno , Wanderley João Luiz , Silva Fabricio R A , Viola João P B , Weber Georg , Castronovo Vincent , Bellahcène Akeila , Gimba Etel R P
Published: August 2014

Introduction: Ovarian carcinoma (OC) is one of the most aggressive gynecological diseases and generally diagnosed at advanced stages. Osteopontin (OPN) is overexpressed in OC and is involved in tumorigenesis and metastasis. Alternative splicing of OPN generates 3 isoforms, OPNa, OPNb, and OPNc. Objective: This study aims to characterize the expression profile and functional role of OPN splicing isoforms (OPN-SI) in OC. Methodology: The expression patterns of OPN-SI and a series of genes involved in cancer pathways were analyzed by real-time PCR. In vitro and in vivo functional assays were performed using the ovarian cancer cell line OvCar-3, stably overexpressing the three OPN-SI. Results: OPNc, but not OPNa and OPNb, was specifically expressed on OC samples. We then demonstrated that OPNc isoform has stimulatory effects on OvCar-3 cell proliferation, migration, invasion and anchorage independent cells growth, besides activating tumor formation in nude mice. We also found that these features are mainly mediated by PI3K/Akt signaling pathway. Moreover, OvCar-3 OPNc-overexpressing cells are able to modulate the expression of 34 genes involved in key cancer pathways. Notably, tumors formed by OPNc-overexpressing cells present high expression levels of typical angiogenic markers, such as VEGF-A, VEGFR-2 and CD34. Based on this observation, we also investigated the molecular mechanisms by which OPNc stimulates angiogenic processes. Our data showed that OPNc overexpression activates VEGF-A expression and secretion, also through the PI3K/Akt pathway. This splicing isoform is also able to activate the expression of c-Fos, c-Jun and phospho-c-Jun. OPNc role on activating the phosphorylation of c-Jun is mediated by integrin receptor, in an RGD-dependent manner. In addition, OPNc-conditioned medium is able to induce HUVEC endothelial cell proliferation, migration and adhesion. Conclusion: Thus, the specific targeting of OPNc and its regulated signaling network could be a putative novel strategy to inhibit key steps involved in OC progression and may represent a putative strategy for developing new therapeutic approaches using OPNc as interesting target for OC treatment strategies.

Citation Format: Tatiana M. Tilli, Kivvi D. Mello, Vanessa Franco, Bruno Robbs, João Luiz Wanderley, Fabricio R A Silva, João P B Viola, Georg Weber, Vincent Castronovo, Akeila Bellahcène, Etel R P Gimba. Osteopontin-c splicing isoform contributes to ovarian cancer progression. [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 396. doi:10.1158/1538-7445.AM2013-396
Abstract 4002: Differential regulation of the ERGF-MAPK pathway by deoxycholic acid (DCA) and Ursodeoxycholic acid (UDCA) in colon cancer.
Authors: Centuori Sara M.
Published: August 2014

Obesity and a western diet are risk factors for the development of colorectal cancer and correlate with high levels of bile acids. Specifically, the secondary bile acid deoxycholic acid (DCA) has been shown to act as a tumor promoter in animal studies. Interestingly, the tertiary bile acid Ursodeoxycholic acid (UDCA) was found to have chemopreventive activity in a clinical trial conducted in 1285 individuals with recently resected colorectal adenomas. The mechanism(s) of DCA's tumor promoting effects and UDCA's chemopreventive activities are unclear, however DCA and UDCA have been previously shown to regulate EGFR-MAPK signaling in a differential manner. Because this mitogenic pathway has been shown to play a role in colon cancer cell proliferation and survival, we hypothesize that DCA's tumor promoting effect may stem from its regulation of this mitogenic pathway. We therefore examined the role of DCA in the regulation of the EGFR MAPK pathway in human colon cancer cells as compared to UDCA. Our findings show that DCA can induce ERK1/2 phosphorylation in an EGFR-independent manner in HT-29 colon adenocarcinoma cells, and that this is at least partially dependent on PI3K activation. We also determined that UDCA pretreatment was able to suppress both EGF and DCA-induced EGFR-MAP signaling. By elucidating the mechanism by which DCA and UDCA differentially regulate colon cancer proliferation and survival, we may be able to skew the signaling in colon cancer cells to favor chemoprevention as opposed to tumorgenesis.

Citation Format: Sara M. Centuori. Differential regulation of the ERGF-MAPK pathway by deoxycholic acid (DCA) and Ursodeoxycholic acid (UDCA) in colon cancer. [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 4002. doi:10.1158/1538-7445.AM2013-4002
Abstract 4008: Effects of a combination of Atorvastatin and Biochanin A on signal transduction and invasiveness of pancreatic cancer cell lines.
Authors: Tadinada Satya Murthy , Lai James C.K. , Bhushan Alok
Published: August 2014

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer related deaths in the United States. Low survival rate associated with this cancer can be attributed to late detection, invasion and metastasis besides chemo-resistance and relapse following surgical resection. Our previous studies demonstrated combination treatment employing Biochanin A with Atorvastatin, an anti-hyperlipidemic drug, was more effective in inhibiting pancreatic cell growth compared to monotherapy. Biochanin A is a naturally occurring isoflavone exhibiting anti-cancer properties in different cancer models. Flow Cytometry studies have demonstrated early apoptosis in AsPC-1 pancreatic cancer cells following a 24-hour exposure to the combination treatment while PANC-1 pancreatic cancer cells did not show significant apoptosis after the treatment for 24 hours and 48 hours. Ongoing studies support the possibility that combination treatment induces cell cycle arrest in PANC-1 cells. We also studied the effects of the combination on invasiveness and on expression of proteins involved in signal transduction pathways in these cell lines. Gelatin zymography invasion assay showed a higher Matrix Metalloproteinase-2 (MMP-2) activity in cultures exposed to Atorvastatin while Biochanin A decreased the effects of atorvastatin induced MMP-2 activity in combination treatment. Combination treatment exerted differential effects on MAPK, PI3K/AKT, mTOR and JAK/STAT pathways in these cell lines. These results may have implications in design of new chemotherapeutic strategies for treating pancreatic cancer.

Acknowledgement

This study was supported by grants from INBRE Program, NIH Grant No. P20 RR016454, Mountain States Tumor and Medical Research Institute and University Research Committee, ISU grant.

Citation Format: Satya Murthy Tadinada, James C.K. Lai, Alok Bhushan. Effects of a combination of Atorvastatin and Biochanin A on signal transduction and invasiveness of pancreatic cancer cell lines. [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 4008. doi:10.1158/1538-7445.AM2013-4008
Abstract 4012: GANT-61 inhibits pancreatic cancer stem cell growth in vitro and in NOD/SCID/IL2R gamma null mice xenograft.
Authors: Fu Junsheng , Rodova Mariana , Roy Sanjit K. , Shankar Sharmila , Srivastava Rakesh K.
Published: August 2014

Multiple lines of evidence suggest that the Sonic Hedgehog (Shh) signaling pathway is aberrantly reactivated in pancreatic cancer stem cells (CSCs). The objectives of this study were to examine the molecular mechanisms by which GANT-61 (Gli transcription factor inhibitor) regulates stem cell characteristics and tumor growth. Effects of GANT-61 on CSC's viability, spheroid formation, apoptosis, DNA-binding and transcriptional activities, and epithelial-mesenchymal transition (EMT) were measured. Humanized NOD/SCID/IL2Rgammanull mice were used to examine the effects of GANT-61 on CSC's tumor growth. GANT-61 inhibited cell viability, spheroid formation, and Gli-DNA binding and transcriptional activities, and induced apoptosis by activation of caspase-3 and cleavage of Poly-ADP ribose Polymerase (PARP). GANT-61 increased the expression of TRAIL-R1/DR4, TRAIL-R2/DR5 and Fas, and decreased expression of PDGFRα and Bcl-2. GANT-61 also suppressed EMT by up-regulating E-cadherin and inhibiting N-cadherin and transcription factors Snail, Slug and Zeb1. In addition, GANT-61 inhibited pluripotency maintaining factors Nanog, Oct4, Sox-2 and cMyc. Suppression of both Gli1 plus Gli2 by shRNA mimicked the changes in cell viability, spheroid formation, apoptosis and gene expression observed in GANT-61-treated pancreatic CSCs. Furthermore, GANT-61 inhibited CSC tumor growth which was associated with up-regulation of DR4 and DR5 expression, and suppression of Gli1, Gli2, Bcl-2, CCND2 and Zeb1 expression in tumor tissues derived from NOD/SCID IL2Rγ null mice. Our data highlight the importance of Shh pathway for self-renewal and metastasis of pancreatic CSCs, and also suggest Gli as a therapeutic target for pancreatic cancer in eliminating CSCs.

Citation Format: Junsheng Fu, Mariana Rodova, Sanjit K. Roy, Sharmila Shankar, Rakesh K. Srivastava. GANT-61 inhibits pancreatic cancer stem cell growth in vitro and in NOD/SCID/IL2R gamma null mice xenograft. [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 4012. doi:10.1158/1538-7445.AM2013-4012
Abstract 4014: The therapeutic potential of ICG-001 and β-catenin specific RNAi in liver cancer development.
Authors: Kassa Anketse D. , Medina Vivian G. , Zeng Ni , He Lina , Nguyen Cu , Smbatyan Goar , Rieger Megan , Kahn Michael , Stiles Bangyan L.
Published: August 2014

Liver cancer is one of the most common malignant tumors. It is reported to be the third most lethal malignancy worldwide. Recent studies including our own identified CD133+ cell population as the tumor initiating cells for liver cancer. Tumor suppressor PTEN (phosphatase and tensin homologue deleted on chromosome ten) is aberrantly expressed in liver cancers. Liver specific Pten (Pm) null mice develop liver cancer following an extensive phase of chronic lipid accumulation and demonstrate escalating levels of hepatic injury markers from 6-12M, prior to hepatic progenitor cell proliferation. Concomitantly, expression of mRNA levels for Wnt ligands and receptors also increased progressively. Wnt/β-Catenin signaling pathway has various roles in regulating embryonic development and tumorigenesis. In Pten null liver progenitor cell line and tissues; we observed high levels of βcatenin, a downstream target component of the Wnt signaling pathway compared to control cells.

In this study, we investigate the role of the Wnt/β-Catenin pathway in the activation of hepatic progenitor cells in the Pten deletion liver cancer model using two methods: a novel Wnt/β-Catenin inhibitor ICG-001 and β-Catenin specific RNAi. ICG-001 is a small molecule, which specifically inhibits β-Catenin/CBP interaction. Coactivator CBP/β-Catenin/T cell factor (TCF) mediated transcription has been reported as critical for stem cell/progenitor cell proliferation.

ICG-001 significantly attenuates the proliferation of hepatic cancer stem cells in vitro and in vivo. Our preliminary results demonstrate inhibition of Wnt/β-Catenin pathway using sh-β-Catenin in vitro can effectively inhibit the proliferation of CD133+ hepatic cancer stem cells.

These observations indicate that Wnt/β- Catenin pathway likely mediates proliferation of hepatic cancer stem cell. Targeting this pathway using ICG-001 and/or β-Catenin directed shRNA holds promise for the treatment and eradication of liver cancer.

Citation Format: Anketse D. Kassa, Vivian G. Medina, Ni Zeng, Lina He, Cu Nguyen, Goar Smbatyan, Megan Rieger, Michael Kahn, Bangyan L. Stiles. The therapeutic potential of ICG-001 and β-catenin specific RNAi in liver cancer development. [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 4014. doi:10.1158/1538-7445.AM2013-4014
Abstract 4020: Genome-wide analysis of stepwise hepatocarcinogenesis using next generation sequencer.
Authors: Midorikawa Yutaka , Takayama Tadatoshi , Yamamoto Shogo , Ueda Hiroo , Sonoda Kotaro , Takane Kiyoko , Tsuji Shingo , Nagae Genta , Tatsuno Kenji , Morgan Margaret , Wheeler David , Aburatani Hiroyuki
Published: August 2014

Early hepatocellular carcinoma (HCC) originates from the hepatocytes of chronic liver disease and develops into classical HCC. To identify sequential genomic changes in multistep hepatocarcinogenesis, we analyzed fusion genes and mutations using next generation sequencer about 40 early and 80 advanced HCCs. First, exome sequence showed that base sequence aberrations were more frequent in advanced HCC than early HCC, and mutations including TP53 (24%), CTNNB1 (24%), and ARID1A (16%) were recurrently observed in both early and advanced HCCs. Consistent with our previous report homozygous deletions near CSMD1 and CDKN2A were also found in 8 and 5 cases, respectively, only in advanced HCC. Next, we detected 4 types of fusion gene, including interchromosomal (translocation), intrachromosomal (deletion), intrachromosomal (translocation), and inversion by RNA sequencing. We also found that every HCC harbored 2 to 5 fusion genes, which were more frequent in advanced than early HCC, although there were no recurrent rearrangements in neither advanced nor early HCCs. Especially chromathripsis, in which 10 to 100 rearrangements were localized in the specific genomic regions and genomic features imply chromosome breaks occur in one-off crisis, was the event observed only in advanced HCC. Taken together, genomic changes were more frequent in advanced HCC, suggesting early HCC originates in the chronic liver disease and progresses into advanced HCC with genomic aberrations.

Citation Format: Yutaka Midorikawa, Tadatoshi Takayama, Shogo Yamamoto, Hiroo Ueda, Kotaro Sonoda, Kiyoko Takane, Shingo Tsuji, Genta Nagae, Kenji Tatsuno, Margaret Morgan, David Wheeler, Hiroyuki Aburatani. Genome-wide analysis of stepwise hepatocarcinogenesis using next generation sequencer. [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 4020. doi:10.1158/1538-7445.AM2013-4020
Abstract 4025: Data-driven computational modeling to identify biomarkers of response to lenvatinib (E7080) in melanoma.
Authors: Kadowaki Tadashi , Funahashi Yasuhiro , Matsui Junji , Pavan Kumar , Sachdev Pallavi , O'Brien Jim , Xing Heming , McDonagh Paul D. , Khalil Iya , Kurzrock Razelle , Hong David S. , Nemunaitis John
Published: August 2014

Background: Lenvatinib is an oral tyrosine kinase inhibitor targeting VEGFR1-3, FGFR1-4, RET, KIT and PDGFRβ. Anti-tumor activity has been observed in melanoma patients in Phase I studies. We applied an integrative supercomputer-driven analysis approach to identify biomarkers of lenvatinib treatment response in melanoma patients.

Methods: Clinical data sets including tumor response data (RECIST), progression free survival (PFS), pharmacokinetic parameters (PK) and molecular data sets including baseline tumor gene expression (Affymetrix U133Plus2) and BRAF and NRAS mutational status, were collected from 18 patients with metastatic melanoma who received lenvatinib 10 mg orally twice daily in 28-day cycles. These clinical and molecular data sets were used to generate computational models developed using REFS (Reverse Engineering and Forward Simulation) modeling platform, which utilizes Bayesian network inference and simulations. Simulations were performed to identify biomarkers of lenvatinib treatment response. These potential predictive biomarkers were then tested for their ability to predict response to lenvatinib in preclinical models.

Results: Using a model comprising gene expression, mutational status and PK data, REFS identified a panel of 18 potential predictive biomarkers of lenvatinib treatment response. Identified biomarkers were able to predict up to 89% of the observed variance in the tumor response data. A total of 32 identified genes including 6 candidate predictive biomarkers (TARBP2, CACNA1, C7ORF, RAP2A, SHMT1, IL22RA2) were further validated in a preclinical melanoma model system (n=12) and tissue bank samples with matched normal adjacent tissue (n=21). Expression of 14 genes correlated with relative tumor volume (r>0.35 or r<-0.35) in the preclinical model and expression levels of 19 genes were found to be higher in tumor compared to normal tissue (FC>2).

Conclusions: Potential predictive biomarkers of lenvatinib treatment response in melanoma patients were identified by computational modeling and validated in a preclinical model system and tumor tissue bank samples. The identified biomarkers will be tested for their predictive value in an ongoing Phase 2 trial.

Citation Format: Tadashi Kadowaki, Yasuhiro Funahashi, Junji Matsui, Kumar Pavan, Pallavi Sachdev, Jim O'Brien, Heming Xing, Paul D. McDonagh, Iya Khalil, Razelle Kurzrock, David S. Hong, John Nemunaitis. Data-driven computational modeling to identify biomarkers of response to lenvatinib (E7080) in melanoma. [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 4025. doi:10.1158/1538-7445.AM2013-4025
Abstract 4033: Gene expression of EdU proliferating cells made possible with improved click chemistry.
Authors: Clarke Scott T. , Cheng Angie , Rukavishnikov Aleksey , Yan Michelle , Singh Upinder , Gee Kyle , Magdaleno Susan
Published: August 2014

Gene expression profiling of cancer cells has become increasingly common over the past five years. Especially important is identifying changes in gene expression caused by drug treatment leading to side populations with altered pathways. These populations can escape drug treatment and lead to recurrence. Characterizing gene expression of proliferating cells requires a method of identifying proliferation while simultaneously maintaining high quality RNA. Typically, cells are screened in a drug panel in the presence of tritiated thymidine or BrdU to identify the drug resistance in the proliferating population. Proliferation detection with these methods is neither rapid nor does it yield high quality RNA for profiling. A new proliferation method based on click chemistry using an alkyne modified thymidine analog (EdU), which can be used to rapidly identify replicating cells appears ideally suited for this application; however, copper used for the catalysis of the click reaction causes undesired degradation of RNA. Recent improvements to the copper based click reaction are presented here whereby the copper is tightly controlled, resulting in higher quality RNA useful for sequencing.

In this study we demonstrate gene expression changes in the proliferating population of a drug resistant cancer cell line compared to the parental cell lines. Proliferating cells were identified using an improved click reaction comprising of a copper(I) ligand combined with a picolyl modified dye azide detection reagent. Total RNA from these cells was isolated and gene expression was assessed by targeted RNA sequencing using an RNA Apoptosis panel and an RNA Cancer panel using proton based sequencing technology.

Use of an improved copper “safe” click reaction for detection of nascent DNA, allows for detection of incorporated click analog for measuring proliferation and subsequent isolation of high quality RNA for use in sequence analysis.

Citation Format: Scott T. Clarke, Angie Cheng, Aleksey Rukavishnikov, Michelle Yan, Upinder Singh, Kyle Gee, Susan Magdaleno. Gene expression of EdU proliferating cells made possible with improved click chemistry. [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 4033. doi:10.1158/1538-7445.AM2013-4033