bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A21: Human breast-to-brain metastases display GABAergic properties in the neural niche
Authors: Neman Josh , Termini John , Kowolik Claudia M. , Hambrecht Amanda C. , Jandial Rahul , Roberts Eugene
Published: August 2014

The high frequency and poor prognosis of breast-to-brain metastasis (BBM) indicates the need for detailed studies of metastatic breast cancer cells, the brain microenvironment, and the interactions between the two that lead to breast cancer colonization and proliferation. Because neurotransmitters are a major component of the brain microenvironment, we examined fresh tissue and cells from recent surgical specimens of BBM to determine if they expressed CNS-specific neurotransmitter characteristics. We found that BBM cells expressed higher levels of the classical neurotransmitter receptors relative to primary breast cancer, and that, overall, they had a GABAergic phenotype similar to that of neuronal cells. Specifically, GABAAR, GAD67, GABA transporter, and GABA transaminase were highly expressed in BBM cells indicating that they utilize GABA. Moreover, we determined BBM cells exploit the GABA shunt in order to enhance energy production and proliferative potential in vitro. BBM cells also expressed the GABAergic interneuron-specific proteins, parvalbumin and reelin. Parvalbumin is a calcium binding protein that is involved in regulating the local inhibitory effects that GABAergic interneurons exert on pyramidal neurons, and reelin is an extracellular matrix glycoprotein that is involved in the migration and positioning of neurons. These data suggest that metastases of mesenchymal origin can assume neural characteristics when exposed to the brain micronenvironment to facilitate colonization.

Citation Format: Josh Neman, John Termini, Claudia M. Kowolik, Amanda C. Hambrecht, Rahul Jandial, Eugene Roberts. Human breast-to-brain metastases display GABAergic properties in the neural niche. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A21.
Abstract A29: Cooption of the reactive stroma by brain metastasis relies on protease inhibitors
Authors: Valiente Manuel , Lee Derek J. , Obenauf Anna , Huse Jason T. , Chaft Jamie E. , Kris Mark G. , Brogi Edi , Massagué Joan
Published: August 2014

Metastatic cancer cells are known to get benefit from the interaction with their microenvironment. The presence of metastatic cells in a target organ induces changes in the stroma. This reactive stroma is very evident in the brain, where cancer cells are ubiquitously associated with reactive glia. Since reactive astrocytes are known sources not only of trophic factors but also deleterious signals, we hypothesized that the ability of metastatic cells to develop in a target organ requires the ability to neutralize these deleterious signals.

We detected reactive astrocytes in the proximity of brain metastasis producing plasminogen activators (tPA and uPA), which activate Plasmin, one of the main proteases in the brain. Plasmin activity in the brain acts as a deleterious factor to metastatic cells in this organ. Cancer cells selected in vivo for their ability to metastasize in the brain overexpress Serine Protease Inhibitors (SERPINs) that block Plasmin activation. Moreover, a significant cohort of human brain metastasis samples from both lung and breast adenocarcinoma scored positive for specific members of the SERPIN family. When the cancer cells were depleted of SERPINs their brain metastatic activity was significantly reduced in all experimental models, including breast and lung adenocarcinoma xenografts and syngeneic mouse models. Brain metastatic cells are sensitive to Plasmin activated killer cytokines (sFASL), and their ability to survive and to grow in the brain depends on genes whose products are inactivated by Plasmin cleavage (VEGFA and L1CAM).

The cooption of SERPINs by brain metastatic cells is required to avoid the deleterious consequences of the reactive stroma and subsequently benefit from the interaction with the microenvironment.

Citation Format: Manuel Valiente, Derek J. Lee, Anna Obenauf, Jason T. Huse, Jamie E. Chaft, Mark G. Kris, Edi Brogi, Joan Massagué. Cooption of the reactive stroma by brain metastasis relies on protease inhibitors. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A29.
Abstract A32: Colorectal cancer stroma: Tumor cells in disguise
Authors: Celesti Giuseppe , Caro Giuseppe Di , Bianchi Paolo , Grizzi Fabio , Basso Gianluca , Doni Andrea , Marchesi Federica , Marra Giancarlo , Roncalli Massimo , Mantovani Alberto , Malesci Alberto , Laghi Luigi
Published: August 2014

Background: Epithelial-to-mesenchymal transition (EMT) is thought to mediate cancer cell dissemination and metastasis. However, the role of EMT in the initial progression of human cancer remains unproven, since delaminating neoplastic cells have never been identified in the peritumoral stroma.

Aim: To demonstrate the existence of EMT-neoplastic cells in mesenchymal disguise in the stroma of human colorectal cancer (CRC).

Methods and Materials / Patients: In vitro, gene expression profile analysis of 18 CRC cells by microarray. Transfection/silencing of TWIST1 in CRC cells and invasion and migration assay. Immunohistochemistry for TWIST1 of 11 adenomas and of 201 CRC specimens. Combined TWIST1 immunofluorescence and in situ hybridization (iFISH) for CEP7 and 20 of 20 CRC specimens. In vivo, orthotopic graft of murine CRC cells CT26 in the rectal sub-mucosa of Balb/c mice.

Results: TWIST1 was necessary to maintain mesenchymal phenotype and invasiveness of microsatellite-stable (MSS) cellular models of EMT, either spontaneous (CoLo741) or induced by transfection (SW480). TWIST1 mRNA was not translated in MSI CRC cells (HCT116). In an orthotopic mouse model of CRC, syngenic TWIST1-positive cells (CT26) acquired a mesenchymal phenotype when invading the peritumoral tissues. In human CRC tissues, the presence of TWIST1-positive cells within the stroma correlated with tumor MSS status (p=0.05), stage IV (p=0.02), and patient prognosis (DSS, p<0.01). Trisomies of chromosome 7 and/or chromosome 20 were detected within the tumor compartment of 17/20 CRC specimens. In each of these 17 tumors, TWIST1-positive cells with matching chromosomal gains were traceable in the peritumoral stroma (86 of 776 counted cells, 11.1%), whereas no trisomy was recognized in TWIST1-negative stromal cells (0 of 1249 cells; p<0.001).

Conclusion: TWIST1 plays a crucial role in maintaining EMT in human CRC. By using TWIST1 as marker of EMT cells, we demonstrated that stromal cells, disguised as fibroblasts, are actually neoplastic. Targeting TWIST1+ stromal cells might open new scenarios for interfering with the metastatic cascade of CRC.

Note: This abstract was not presented at the conference.

Citation Format: Giuseppe Celesti, Giuseppe Di Caro, Paolo Bianchi, Fabio Grizzi, Gianluca Basso, Andrea Doni, Federica Marchesi, Giancarlo Marra, Massimo Roncalli, Alberto Mantovani, Alberto Malesci, Luigi Laghi. Colorectal cancer stroma: Tumor cells in disguise. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A32.
Abstract A53: CXCR4 drives the metastatic phenotype in breast cancer through induction of CXCR2, transactivation of HER2/EGFR, and activation of MEK and PI3K pathways
Authors: Sobolik Tammy , Su Yingjun , Wells Sam , Richmond Ann
Published: August 2014

Introduction: Aberrant expression of CXCR4 in human breast cancer correlates with metastasis and poor prognosis. Expression of a carboxyl-truncated CXCR4 (CXCR4ΔCTD) is associated with constitutive CXCR4 activity in MCF-7 breast cancer cells, increased motility, loss of E-cadherin, enhanced estrogen independent growth and lung metastasis in vivo. Here, we characterize the mechanism by which CXCR4 induces a mesenchymal phenotype with metastatic properties.

Methods: MCF-7 cells expressing wild-type CXCR4 (CXCR4WT) or CXCR4ΔCTD were evaluated in two-dimensional (2D) and three-dimensional reconstituted basement membrane (3D rBM) cultures for cell morphology and expression of factors that regulate the epithelial to mesenchymal transition (EMT) and cell migration. Effects of inhibition of key cytokine/chemokine signaling pathways activated by CXCR4 were examined. Tumor cell migration within the tumor microenvironment was followed using intravital imaging.

Results: In 2D culture, MCF-7 CXCR4ΔCTD but not CXCR4WT cells, exhibited EMT characterized by up-regulation of ZEB-1 and cadherin 11, loss of E-cadherin, p120 isoform switching, and activation of ERK1/2, and MMP-2. The up-regulation of ZEB-1 was coincident with down-regulation of miR-200b and miR200c. In contrast to the 2D environment, 3D rBM MCF-7 CXCR4WT cultures exhibited the same stellate morphology of CXCR4ΔCTD cells, while MCF-7 control cells formed round acinar clusters. Expression of CXCR4 resulted in induction of expression of CXCR2, CXCR7, CXCL1, CXCL8, CCL2, IL-6 and GM-CSF by MCF-7 cells in 3DrBM, but not 2D culture. CXCR4 expression also induced transactivation of HER2 and EGFR. CXCL1, CXCL8 and CXCR2 appear to signal through a CXCR2 autocrine loop. In 3D rBM culture combined inhibition of CXCR4 and MEK1, or PI3K and MEK1/2 resulted in a change in morphology of CXCR4 expressing cells from stellate to round acinar clusters resembling controls. Combined inhibition of CXCR2 with MEK1, PI3K, or CXCR4 inhibitors also resulted in reversion of the stellate phenotype in 3D rBM. In vivo studies revealed that MCF-7 CXCR4WT and MCF-7CXCR4ΔCTD tumor cells migrate in a collective manner capable of lymphatic spread in athymic nude mice.

Conclusions: Elevated expression or constitutive activation of CXCR4 results in EMT that is accompanied by up-regulation of receptors and cytokines important in collective cell migration leading to lymphatic invasion and tumor metastasis.

Citation Format: Tammy Sobolik, Yingjun Su, Sam Wells, Ann Richmond. CXCR4 drives the metastatic phenotype in breast cancer through induction of CXCR2, transactivation of HER2/EGFR, and activation of MEK and PI3K pathways. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A53.
Abstract A61: Lysophosphatidic acid induces ovarian cancer cell invasion through EGFR phosphorylation and proteolytic enzyme expression
Authors: Jeong Kang Jin , Cho Kyung Hwa , Kim Jeong Jin , Choi Moon Jung , Lee Hoi Young
Published: August 2014

Lysophosphatidic acid (LPA) is a biolipid that augments proliferation and metastasis of various cancer cells. LPA was first implicated in human oncogenesis, as higher levels of LPA was observed in the ascitic fluid of ovarian cancer patient than healthy subjects. In the present study, we elucidate the underlying mechanisms by which LPA induces proteolytic enzyme expression and ovarian cancer progression. LPA induced MMP-9 expression in CAOV-3 and PA-1 cells and uPA expression in SKOV-3 cells. LPA-induced proteolytic enzyme expression was required for the invasion of ovarian cancer cells expressing corresponding enzymes. Further we observed that both Gi and G13 contribute to LPA-induced EGFR activation. The activated EGFR in turn initiates a Ras/Rho/ROCK signaling cascade, leading to proteolytic enzyme secretion. Moreover, we provide evidence that resveratrol inhibits EGFR phosphorylation and subsequent activation of a Ras/Rho/ROCK signaling. Therefore, we demonstrate a mechanistic cascade of LPA activating EGFR through Gi and G13 thus inducing a Ras/Rho/ROCK signaling for proteolytic enzyme expression and ovarian cancer cell invasion, as well as interference of the cascade by resveratrol through blocking EGFR phosphorylation. [Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2011-0015761 and 2010-0022216)]

Citation Format: Kang Jin Jeong, Kyung Hwa Cho, Jeong Jin Kim, Moon Jung Choi, Hoi Young Lee. Lysophosphatidic acid induces ovarian cancer cell invasion through EGFR phosphorylation and proteolytic enzyme expression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A61.
Abstract A8: Fibulin-5 (FBLN5) initiates EMT and induces MMPs expression via β1 integrin/EGFR/ERK pathway
Authors: Lee Yonghun , Schiemann William P.
Published: August 2014

Epithelial-mesenchymal transition (EMT) is a normal physiological process that regulates tissue development, remodeling and repair; however, aberrant EMT also elicits disease development in humans, including lung fibrosis, rheumatoid arthritis and cancer cell metastasis. Transforming growth factor-β (TGF-β) is a master regulator of EMT in normal mammary epithelial cells (MECs), wherein this pleiotropic cytokine also functions as a potent suppressor of mammary tumorigenesis. In contrast, malignant MECs typically become resistance to TGF-β-mediated cytostasis and develop the ability to proliferate, invade and metastasize when stimulated by TGF-beta. Therefore, the understanding of how TGF-β promotes EMT may offer new insights into targeting the oncogenic activities of TGF-β in human breast cancers. Previously, we showed that the TGF-β gene target, FBLN5, initiates EMT and enhances that induced by TGF-β. FBLN5 stimulated matrix metalloproteinase (MMP 2,3, and 9) expression and activity, leading to MEC invasion and EMT. While WT FBLN5 induces EMT and MMP expression, RGE mutant (loss of integrin binding activity) of FBLN5 fails to induce EMT and MMP expression. We show here that FBLN5 induces EGFR expression, and the inhibition of EGFR and ERK1/2 MAPK with AG 1478 β3 integrin suppresses FBLN5 induced MMP expression, the expression of D119A mutant β3 integrin fails to suppress FBLN5-induced MMP expression. In addition, knock-down of β1 integrin reverses FBLN5-induced EMT and suppress FBLN5-induced MMPs expression. These results suggest FBLN5 induces EMT and MMP expression in β1 integrin-dependent β1 integrin/EGFR/ERK signaling pathway in MECs. These findings identify a novel EMT and tumor-promoting function for FBLN5 in developing and progressing breast cancers.

Citation Format: Yonghun Lee, William P. Schiemann. Fibulin-5 (FBLN5) initiates EMT and induces MMPs expression via β1 integrin/EGFR/ERK pathway. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A8.
Abstract A92: Killing quiescent cancer cells in vitro and in vivo with tumor targeting Salmonella typhimurium A1-R
Authors: Yano Shuya , Zhang Yong , Uehara Fuminari , Hiroshima Yukihiko , Miwa Shinji , Hoffman Robert M. , Zhao Ming
Published: August 2014

Quiescent cancer cells are resistant to cytotoxic agents which target only proliferating cancer cells. Cancer cells were imaged to indicate the cell-cycle phase of each cancer cell in real time in solid tumors growing in vivo as well for cancer cells as in vitro using a fluorescence ubiquitination cell cycle indicator (FUCCI). With FUCCI, quiescent cancer cells express mKusabira-Orange fluorescent protein (red) and proliferating cells express mAzami-Green fluorescent protein (green). FUCCI-labeled cancer cells in monolayer culture and tumor spheres were treated with Salmonella typhimurium A1-R. Time-lapse confocal imaging showed that A1-R encircled and invaded quiescent cancer cells in monlayer culture, thereby eliminating them. Cancer cells in tumor spheres in serum-free culture become and remain quiescent. A1-R infected and killed quiescent cancer cells in tumor spheres. In contrast, cytotoxic agents did not kill quiescent cancer cells in tumor spheres. A1-R infection of FUCCI-expressing subcutaneous tumors growing in nude mice resulted in killing quiescent cancer cells resistant to cytotoxic agents. This study demonstrates that A1-R can kill quiescent cancer cells and suggest a new therapeutic paradigm potentially more effective than current therapy of cancer.

Citation Format: Shuya Yano, Yong Zhang, Fuminari Uehara, Yukihiko Hiroshima, Shinji Miwa, Robert M. Hoffman, Ming Zhao. Killing quiescent cancer cells in vitro and in vivo with tumor targeting Salmonella typhimurium A1-R. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A92.
Abstract A95: Therapeutic effects of standardized Wedelia chinensis extract on prostate cancer in vivo
Authors: Hsiao Pei-Wen Hsiao , Tsai Chin-Hsien , Yang Yu-Chih Hsiao , Lee Ming-Ting
Published: August 2014

Globally, prostate cancer is the sixth leading cause of cancer-related death in men with higher prevalence in Western countries. Signaling pathway of androgen receptor plays a central role in the prostate cancer progression. Accordingly, approaches and molecular mechanisms to ablate the androgen receptor function in prostate cancer have been actively studied to improve the disease treatment. However, the disease inevitably develops resistance to all forms of hormonal therapies. To mimic the clinical progression of human prostate cancer in mouse model, we implant human prostate cancer cell lines that were tagged with Luc2 reporter by lentivirus-mediated infection in the prostate gland of nude mouse. After tumor established, the orthotopic xenograft were treated with castration and progressed to castration-resistant prostate cancer (CRPC). While chemotherapy of the CRPC with intravenous docetaxel induced initial tumor regression, the tumors inevitably recurred with subsequent metastasis. We prepared a standardized herbal medicine from Wedelia chinensis plant with compounds that attenuate prostate cancer growth in vivo. Here, we investigated the combinational effect of the herbal remedy and docetaxel in the treatment of CRPC. In contrast to the docetaxel monotherapy, the docetaxel plus herbal remedy significantly delayed the tumor recurrence at primary site and distant sites, among which the CRPC in 27% mice was cured. The promising therapeutic effects of candidate herbal medicine against locally advanced tumor and subsequent metastases warrant further analyses for the underlying cellular and molecular mechanisms. (Supported by Academia Sinica, Career Development Award)

Citation Format: Pei-Wen Hsiao Hsiao, Chin-Hsien Tsai, Yu-Chih Hsiao Yang, Ming-Ting Lee. Therapeutic effects of standardized Wedelia chinensis extract on prostate cancer in vivo. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A95.
Abstract B101: NFAT regulates a gene expression program associated with invasiveness and poor prognosis in colorectal cancer
Authors: Tripathi Manish K. , Mima Shinji , Shi Zhiao , Prodduturi Naresh , Jing Zhu , Ciombor Kristen K. , Chen Xi , Deane Natasha , Beauchamp Robert D. , Zhang Bing
Published: August 2014

Colorectal cancer is the second leading cause of cancer-related death in the United States. In order to understand the regulatory mechanisms underlying poor prognosis in colorectal cancer, we analyzed fourteen human colorectal cancer microarray data sets and identified co-expressed modules using network analysis. We next filtered these modules using gene expression data from a mouse model of metastatic colon cancer, narrowing down to a candidate metastasis-related module, and identified NFAT as its potential transcriptional regulator. The NFAT family and their identified targets were found to be upregulated in human colorectal cancer patients. Analysis of NFAT family members expression in mouse and human microarray datasets, revealed NFATc1 to be differentially expressed between metastatic and non-metastatic, and between disease progression and no disease progression, respectively. We found that high NFATc1 expression correlated with significantly increased invasion (p<0.0001) and migration (p<0.005) in mouse colon cancer cells. We show that RNAi- based knockdown of NFATc1 and functional inhibition by the calcineurin inhibitor FK506 resulted in downregulation of predicted NFAT target genes from the metastatic module and decreased cancer cell invasiveness. Finally, we showed that the expression of NFAT target genes was significantly correlated with both disease-specific and disease-free survival in Stage II and III colorectal cancer patients. Our studies suggest a role for NFATs in colon cancer cell invasion and a potential application for the NFAT driven program as a biologically anchored prognostic gene expression signature.

Citation Format: Manish K. Tripathi, Shinji Mima, Zhiao Shi, Naresh Prodduturi, Zhu Jing, Kristen K. Ciombor, Xi Chen, Natasha Deane, Robert D. Beauchamp, Bing Zhang. NFAT regulates a gene expression program associated with invasiveness and poor prognosis in colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B101.
Abstract B12: The role of stromal cells in supporting tumor-initiating cells in serous ovarian carcinoma
Authors: Hussain Ali , Stewart Jocelyn , Hyatt Elzbieta , Neel Benjamin , Ailles Laurie
Published: August 2014

High grade serous ovarian carcinoma has been shown to be a highly heterogeneous disease. In vivo studies demonstrate that the tumor initiating frequency (TIF) varies substantially from case to case. Nonetheless, the tumor initiating subset remains a rare population within the epithelial compartment and can be enriched using the cell surface marker CD133 (Stewart et al., PNAS, 2011). The tumor microenvironment may play a role in supporting and maintaining tumor initiating cells (TIC) through direct and/ or indirect cross talk. The contribution of the mesenchymal component of the microenvironment may prove to be essential in providing such support as has been previously shown in studies on breast and prostate cancers. We hypothesize that cancer associated fibroblasts (CAFs) serve as a niche that supports and maintains the tumorigenic potential of TICs in SOC.

We have derived and established CAF lines from bulk primary tumors and characterized their phenotype through immunostaining. Those lines stained positive for mesenchymal markers (Vimentin and Alpha-SMA), and stained negative for the epithelial marker Cytokertain. A profile of surface markers expressed on the surface of these CAF lines was then generated by running a flow cytometry-based high throughput screen (HTS) for 370 known cell surface proteins. Those markers are being validated for their specificity by immunostaining, FACS, qPCR, and in vivo work. Preliminary data obtained through immunoflourescnce and FACS support the specificity of our candidate stromal marker CD90. Furthermore, FACS data show that CD90 is expressed more brightly on fibroblasts than on epithelial cells (EpCAM+) in bulk SOC cases. Consequently, CD90 has been selected for sorting stromal cells for additional validation. Quantitative PCR analysis of CD90+EpCAM- sorted populations further validates their over-expression of mesenchymal genes when compared to CD90-EpCAM+ sorted populations. Moreover, functional validation of the influence of fibroblasts on the growth of tumor cells is currently being investigated through co-injection and co-culture assays. Preliminary data show that the presence of fibroblasts better supports the growth of epithelial colonies in culture than when compared to other conditions.

Interplay between the niche and a specific subset of epithelial cells may promote those cells to become more tumorigenic. Such an interaction may be dependent on direct physical contact and/or indirect cross talk. Ultimately, we aim on understanding the mechanisms that govern these interactions.

Citation Format: Ali Hussain, Jocelyn Stewart, Elzbieta Hyatt, Benjamin Neel, Laurie Ailles. The role of stromal cells in supporting tumor-initiating cells in serous ovarian carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B12.
Abstract B51: Potential role of CXCR3 in breast cancer bone metastasis
Authors: Feng Wendy , Unlap Tino
Published: August 2014

Breast cancer has a high tendency to metastasize to the bone. Breast cancer bone metastasis causes several serious clinical complications such as extreme bone pain, bone fracture, hypercalcemia, and spinal cord compression. Furthermore, once breast cancer metastasizes to the bone, it often becomes incurable. However, the molecular mechanism of breast cancer bone metastasis is not fully understood. CXCR3 and its ligands (CXCL9, CXCL10 and CXCL11) have been shown to play a role in the initiation and progression of cancer metastasis in various types of cancer including breast cancer. Specifically, it has been demonstrated that CXCR3 can promote breast cancer cell line metastasis to the lung in mouse models. A recent study has revealed that CXCR3 is highly expressed in breast cancer tissues and that its expression is positively associated with breast cancer progression. Since the bone is the most common site of breast cancer metastasis, it is possible that CXCR3 and one or more of its ligands are also involved in breast cancer bone metastasis by promoting extravasation of breast tumor cells to the bone. The objective of this study was to carry out several experiments to test this possibility. First, we assessed whether MDA-MB-231 breast cancer cell line expresses CXCR3 using RT-PCR and Western blot analysis. Our data indicate that both CXCR3 mRNA and protein are highly expressed in MDA-MB-231 cells. Next, the expression of CXCR3 ligands (CXCL9, CXCL10, and CXCL11) in bone cells (RAW264.7 and RAW264.7-derived osteoclasts) was examined using RT-PCR, and the data indicate that the bone cells express CXCL10 and CXCL11. In particular, CXCL11 mRNA expression levels increase during osteoclast differentiation. Finally, we performed chemotaxis assays to determine whether the bone cells are able to induce directed migration of MDA-MB-231 cells. We found that the bone cells can exert profound chemotactic effects on MDA-MB-231 cells. Taken together, these findings support the possibility that CXCR3 and its ligands may be involved in breast cancer bone metastasis by mediating breast cancer cell extravasation into the bone marrow. Future studies are needed to address whether CXCR3 is the predominant factor in mediating bone cell-dependent breast cancer migration. Moreover, animal model studies are also needed to investigate whether CXCR3 and its ligands are involved in breast cancer bone metastasis in vivo. If CXCR3 is in fact a predominant factor mediating breast cancer metastasis, CXCR3 may be a potential therapeutic target of breast cancer metastasis.

Citation Format: Wendy Feng, Tino Unlap. Potential role of CXCR3 in breast cancer bone metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B51.
Abstract B72: CX3CR1, a fractalkine receptor, can regulate peritoneal dissemination of epithelial ovarian cancer cells
Authors: Xie Jia , Gurler Hilal , Burdette Joanna , Barbolina Maria
Published: August 2014

Epithelial ovarian carcinoma (EOC) is a leading cause of death from gynecologic malignancies mainly due to metastasis. EOC metastases spread by shedding the malignant cells off from the ovarian surface and seeding to organs and tissues in the peritoneal cavity. The current standard of care, a combination of chemotherapy and surgery, fails to keep patients in remission. Thus, mechanistic understanding of the biology of EOC metastasis and identification of novel targetable molecules are crucial for the development of improved therapeutic approaches. Fractalkine receptor (CX3CR1) belongs to a chemokine family of G-protein coupled receptors. CX3CR1 is expressed by EOC cells and promotes cell migration and proliferation. CX3CR1-positive EOC cells have been shown to adhere to the CX3CL1-positive peritoneal mesothelial cells in vitro. Thus, we hypothesized that CX3CL1/CX3CR1 axis could be important for peritoneal dissemination of metastatic EOC.

In order to test this hypothesis, we performed short-term ex vivo and in vivo adhesion assays, as well as long-term metastasis formation assay. We found that EOC cells prefer omentum to other peritoneal tissues during the short-term in vivo and ex vivo adhesion. Importantly, reduction of CX3CR1 expression in EOC cells resulted in significantly reduced peritoneal adhesion. Furthermore, reduced adhesion correlated with the ability of EOC cells to form overt metastasis. Microenvironmental milieu has been histologically evaluated for stromal cells in the omentum expressing CX3CL1. Taken together, our data indicate that fractalkine signaling is one of the central regulators of EOC metastasis.

Citation Format: Jia Xie, Hilal Gurler, Joanna Burdette, Maria Barbolina. CX3CR1, a fractalkine receptor, can regulate peritoneal dissemination of epithelial ovarian cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B72.
Abstract B86: Ang-2-VEGF-A CrossMab, a novel bispecific human IgG1 antibody blocking VEGF-A and Ang-2 functions simultaneously, mediates potent antitumor, antiangiogenic, and antimetastatic efficiency
Authors: Lorenzon Erica , Kienast Yvonne , Raemsch Romi , Scheuer Werner , Herting Frank , Bernicke Dirk , Schaefer Wolfgang , Regula Joerg , Stubenrauch Kay , Yu Sidney , The Huynh Hung , Martarello Laurent , Klein Christian , Thomas Markus
Published: August 2014

Purpose: VEGF-A blockade has been validated clinically as a treatment for human cancers. Angiopoietin-2 (Ang-2) expression has been shown to function as a key regulator of tumor angiogenesis and metastasis.

Experimental Design: We have recently described a novel generic method for the production of bivalent bispecific human IgG1 antibodies. Subsequently, we have applied the CrossMab technology for the generation of a bispecific antibody recognizing VEGF-A with one arm based on Bevacizumab, and the other arm recognizing Ang-2 based on LC06, a highly Ang-2 selective human IgG1 antibody. Ang-2-VEGF CrossMab was evaluated in vitro and in vivo using molecular, biochemical, cellular and histological analyses to determine its pharmacologic properties, along with its mechanism of action.

Results: Ang-2-VEGF-A CrossMab showed potent tumor growth inhibition in a panel of orthotopic and subcutaneous syngeneic mouse tumors and patient and cell line-derived human tumor xenografts at both early and late stages of tumor development. Furthermore, a strong inhibition of angiogenesis and an enhanced vessel normalization phenotype were observed. In contrast to Ang-1 inhibition, anti-Ang-2-VEGF-A treatment did not aggravate the adverse effect of anti-VEGF treatment on physiological vessels. Moreover, treatment with Ang-2-VEGF-A CrossMab resulted in inhibition of hematogenous spread of tumor cells to other organs and reduced micrometastatic growth in the adjuvant setting.

Conclusion: Our data indicate that Ang-2-VEGF-A CrossMab mediates potent anti-tumor, anti-angiogenic, and anti-metastatic efficacy and represents a promising therapeutic agent for the therapy of cancer.

Citation Format: Erica Lorenzon, Yvonne Kienast, Romi Raemsch, Werner Scheuer, Frank Herting, Dirk Bernicke, Wolfgang Schaefer, Joerg Regula, Kay Stubenrauch, Sidney Yu, Huynh Hung The, Laurent Martarello, Christian Klein, Markus Thomas. Ang-2-VEGF-A CrossMab, a novel bispecific human IgG1 antibody blocking VEGF-A and Ang-2 functions simultaneously, mediates potent antitumor, antiangiogenic, and antimetastatic efficiency. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B86.
Abstract B90: Hypoxia-induced pathological angiogenesis mediates tumor cell dissemination, invasion, and metastasis in a zebrafish tumor model
Authors: Rouhi Pegah , Lee Samantha Lin , Jensen Lasse Dahl , Zhang Danfang , Ji Hong , Hauptmann Giselbert , Ingham Philip , Cao Yihai
Published: August 2014

Mechanisms underlying pathological angiogenesis in relation to hypoxia in tumor invasion and metastasis remain elusive. Here, we have developed a zebrafish tumor model that allows us to study the role of pathological angiogenesis under normoxia and hypoxia in arbitrating early events of the metastatic cascade at the single cell level. Under normoxia, implantation of a murine T241 fibrosarcoma into the perivitelline cavity of developing embryos of transgenic fli1:EGFP zebrafish did not result in significant dissemination, invasion, and metastasis. In marked contrast, under hypoxia substantial tumor cells disseminated from primary sites, invaded into neighboring tissues, and metastasized to distal parts of the fish body. Similarly, expression of the hypoxia-regulated angiogenic factor, vascular endothelial growth factor (VEGF) to a high level resulted in tumor cell dissemination and metastasis, which correlated with increased tumor neovascularization.

Inhibition of VEGF receptor signaling pathways by sunitinib or VEGFR2 morpholinos virtually completely ablated VEGFinduced tumor cell dissemination and metastasis. To the best of our knowledge, hypoxia- and VEGF-induced pathological angiogenesis in promoting tumor dissemination, invasion, and metastasis has not been described perviously at the single cell level. Our findings also shed light on molecular mechanisms of beneficial effects of clinically available anti-VEGF drugs for cancer therapy.

Citation Format: Pegah Rouhi, Samantha Lin Lee, Lasse Dahl Jensen, Danfang Zhang, Hong Ji, Giselbert Hauptmann, Philip Ingham, Yihai Cao. Hypoxia-induced pathological angiogenesis mediates tumor cell dissemination, invasion, and metastasis in a zebrafish tumor model. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B90.
Abstract B92: Identifying different functional mechanisms of invasion from mesenchymal and proneural subtypes of glioblastoma
Authors: Jung Sungwon , Kim Seungchan , Kiefer Jeff , Berens Michael E.
Published: August 2014

This abstract is being presented as a short talk in the scientific program. A full abstract is printed in the Proffered Abstracts section (PR12) of the Conference Proceedings.

Citation Format: Sungwon Jung, Seungchan Kim, Jeff Kiefer, Michael E. Berens. Identifying different functional mechanisms of invasion from mesenchymal and proneural subtypes of glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B92.
Abstract B98: Promoter methylation status of tumor suppressor genes and an inflammatory-related COX-2 gene in cervical intraepithelial neoplasia
Authors: Lin Sheng Hsiung , Yu Chun Chieh , Wu Ming Tsang , Li Ruei Nian
Published: August 2014

Cervical cancer is one of the most common cancer among women. Early detection and prompt treatment would be the best management options. DNA methylation is one of the epigenetic regulation mechanisms leading to gene silencing in neoplastic cells. Silencing of tumor suppressor gene resulted from aberrant methylation has been detected in a majority of human cancers. Here, we applied methylation-specific polymerase chain reaction (MSP) to examine the methylation state of three tumor suppressor genes (rar-beta, p16 and cdh1) and an inflammatory-related cox-2 gene in different stages of cervical intraepithelial neoplasia (CIN). Our analyses revealed that cox-2 gene is in unmethylated form from CIN I to carcinoma specimens. Besides, no significant increase in methylation levels of rar-beta and cdh1 genes was observed. However, the high frequency of aberrant hypermethylation of p16 gene (13.2% in normal specimens ; 18.2% in CIN I; 35.7% in CIN II; 31.6% in CIN III and 15.4% in carcinoma) suggesting that p16 is progressively increased during the development of malignant stages in cervical intraepithelial neoplasia, especially in the absence of HPV infection. The result of bisulfite sequencing indicated that the 10 CpG sites of p16 gene are all methylated in ten individuals. In conclusion, this study identifies promoter methylation analysis of p16 on cervical cell specimens may be an additional tool for current cervical cytomorphology based screening.

Citation Format: Sheng Hsiung Lin, Chun Chieh Yu, Ming Tsang Wu, Ruei Nian Li. Promoter methylation status of tumor suppressor genes and an inflammatory-related COX-2 gene in cervical intraepithelial neoplasia. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B98.
Abstract C14: Loss of haptotaxis facilitates invasion in LKB1-deficient melanoma
Authors: Chan Keefe T. , Asokan Sreeja B. , Bo Tao , Berginski Matthew E. , Liu Wenjin , Cochran Shelly D. , Sharpless Norman E. , Bear James E.
Published: August 2014

Germline mutations in the serine/threonine kinase STK11/LKB1 are associated with Peutz-Jehgers Syndrome, which is characterized by hyperpigmentation of the oral mucosa. Inactivating somatic mutations occur in approximately 10-20% of melanomas; however, how the loss of LKB1 facilitates melanoma invasion remains poorly understood. Using cell lines derived from simultaneous activation of KRas and inactivation of LKB1 in melanocytes, we have investigated melanoma migration upon reconstitution with LKB1. Reexpression of LKB1 diminishes migration during wound healing, spheroid outgrowth into 3D collagen, and overall single cell speed in random motility assays. Furthermore, the formation of invadopodia is independent of LKB1 status in both human and mouse melanomas. Interestingly, using microfluidic devices we have found that loss of LKB1 abrogates the ability of cells to respond to gradients of extracellular matrix (haptotaxis) but does not impair their ability to chemotax to EGF. We have also recently developed a model of orthotopic implantation of multicellular tumor spheroids into the dermis of the mouse ear skin and have validated this approach by recapitulating the finding that LKB1 limits tumorigenesis. We are using this model to image local invasion in vivo by multiphoton microscopy and are currently examining the intriguing hypothesis that loss of extracellular matrix sensing is one aspect that contributes to metastatic migration.

Citation Format: Keefe T. Chan, Sreeja B. Asokan, Tao Bo, Matthew E. Berginski, Wenjin Liu, Shelly D. Cochran, Norman E. Sharpless, James E. Bear. Loss of haptotaxis facilitates invasion in LKB1-deficient melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C14.
Abstract C15: CRMP1 functions to suppress epithelial-mesenchymal transition and invasion capacity of prostate cancer cells
Authors: Cai Ganhui , Yu Shan , Ji Yanping , Chan Franky Leung
Published: August 2014

Collapsin response mediator protein 1 (CRMP1) is originally identified and characterized as a cytoplasmic phosphoprotein involved in the Semaphorin 3A (Sema3A)-regulated growth cone collapse during neural development, via its regulation of cytoskeleton dynamics, and then also characterized as an invasion suppressor in lung cancer. Thus, we hypothesize that CRMP1 may be involved in the epithelial-mesenchymal transition (EMT) process and invasion or metastasis via its regulation on cytoskeleton organization in prostate cancer cells. Results of immunohistochemistry study showed that CRMP1 exhibited a reduced expression pattern in high Gleason-scored clinical prostate cancer tissues. In vitro functional studies showed that stable CRMP1-knockdown could induce EMT in prostate cancer cells (DU145), while its ectopic expression could induce the reverse process mesenchymal-epithelial transition (MET) in both DU145 and PC-3 prostate cancer cells, as evidenced by significant change of cellular morphology and altered expression levels of epithelial and mesenchymal markers. Confocal immunofluorescence showed that reorganization of F-actin was seen after shRNA-mediated CRMP1-knockdown in DU145 cells. It was also revealed that CRMP1 overexpression could confer the resistance to TGF-β1- and Snail-induced EMT in prostate cells. Results of CRMP1 promoter-driven luciferase reporter and ChIP assays indicated that Snail was a direct transrepressor of CRMP1 gene, which showed a downregulation pattern in both prostate cancer cells (DU145) and immortalized prostatic epithelial cells (BPH1) with overexpression of Snail. Furthermore, wound healing and Matrigel invasion assays showed that CRMP1 could inhibit cell migration and invasion capacity in DU145 and PC-3 cells. Taken together, our findings suggest that CRMP1 could play a role in prostate cancer invasion and metastasis, via its regulation on EMT and MET process. (This work was supported by a Direct Grant for Research and a RGC-GRF grant 2009/2010)

Citation Format: Ganhui Cai, Shan Yu, Yanping Ji, Franky Leung Chan. CRMP1 functions to suppress epithelial-mesenchymal transition and invasion capacity of prostate cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C15.
Abstract C17: Identifying novel genes critical to invasion in head and neck squamous cell carcinoma
Authors: Jayakar Sangeeta K. , Loudig Olivier D. , Brandwein-Gensler Margaret , Ryung Kim S. , Prystowsky Michael B. , Segall Jeffrey E. , Belbin Thomas J.
Published: August 2014

Oral cavity squamous cell carcinomas (OCSCC) have a poor patient prognosis, which is attributed to their invasive nature. Our goal was to identify specific genes that are important in OCSCC invasion, utilizing genome-wide expression data generated from HNSCC patient tumor samples of varying degrees of invasiveness. Invasion was assessed using the histological parameter pattern of invasion (POI), which describes the manner in which tumor cells infiltrate the normal tissue at the tumor-host interface. POI was used to categorize tumor resections into five types (1-5), type 5 being the most invasive with a “widely dispersed pattern of tumor infiltrate” and correlated significantly with the appearance of local recurrence and decreased overall survival of the patients after surgery. Global gene expression data was acquired from analysis of the surgical resections from 9 OCSCC patient samples collected at Montefiore Medical Center between 2001 and 2008. Tumor cell-enriched cores from formalin-fixed paraffin-embedded (FFPE) samples were used to generate global gene expression data using the Illumina® WG-DASL array to compare gene expression in four tumors that were WPOI type 5 and five tumors that were WPOI type 3. RNA extracted from flash-frozen tumor samples following surgical resection from the same patients was used to obtain global gene expression data using the Illumina® HumanHT-12 v4 Expression BeadChip. Candidate genes were assessed using in vitro invasion assays following siRNA knockdown of specific gene expression using siGENOME smart pools from Dharmacon. When both gene expression datasets were analyzed, we identified 104 genes that were overexpressed at least 1.5-fold in the WPOI type 5 tumors compared to the less invasive WPOI type 3 tumors. Fifty genes were initially tested based on functional criteria. In our initial screen, 16 of these genes showed a significant reduction in tumor cell invasion when expression was knocked down by siRNA in an OCSCC cell line (UMSCC1). These 16 genes are now being tested in other cell lines as well as in our in vivo mouse model to assess their invasive phenotype. This initial screen of global gene expression data in combination with pattern of invasion has revealed multiple novel genes which may play a critical role in OCSCC invasion and warrant further study.

Citation Format: Sangeeta K. Jayakar, Olivier D. Loudig, Margaret Brandwein-Gensler, Kim S. Ryung, Michael B. Prystowsky, Jeffrey E. Segall, Thomas J. Belbin. Identifying novel genes critical to invasion in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C17.
Abstract C2: High-content imaging facilities modeling of cancer epithelial-mesenchymal transition signaling network
Authors: Zhu Xiaoping , Zhen Yin , Zhao Hong , Wong Stephen
Published: August 2014

Background: The epithelial-mesenchymal transition (EMT) is critically involved in tumorigenesis and tumor progression except its fundamental role to development and tissue remodeling. Although the full spectrum of signaling agents that contribute to EMTs of carcinoma cells remains unclear, the interactions and coordination of individual known molecules or pathways that contribute to EMT phenotype as a network are not well studied. The EMT signaling network is complicate in nature, as such, a systems mathematical modeling is especially valuable to help understand and characterize genes that are mediated individually or in combination in the context of relevant signaling pathways, and such models would help to predict feedback loop in the network and provide candidate strategies to control the signaling transduction furthermore guide us make clinic research available after the further validation.

Experimental design and methods: siRNA-mediated knockdown of individual nine EMT-related genes in mesenchymal like cancer cell line (HMLE-Ras-Snail and U87), and epithelial cell lines (HMLE-Ras-vector and MCF10A) were imaged in a time-lapse manner at single cell resolution. Multiple cell phenotype features were extracted by image informatics analysis. Thirteen proteins including the 9 siRNA targeted genes as well as 4 epithelial and mesenchymal marker proteins were profiled by Western blot. Bioinformatics modeling was performed to create a gene EMT network and describe the genes or pathway interactions which are dominant for EMT or MET phenotypes.

Results: Each individual siRNA knockdown indicated by western blot, concomitantly incucyte captured clear cell morphology changes due to RNA silence: silencing TJP1, PXN, FLNA, CCNE1switched cells to mesenchymal-like, silencing Gli3, CTNNB1, PSEN1 induced cells more Epithelial-like. However not every maker protein showed as significant changes as images parameters (cell width/length ratio, protrusion ect) did. Therefore by using traditional biochemical and imaging two approaches, we will identify the confident genes for EMT regulation.

Conclusion: Our results demonstrate that high-content imaging is a reliable and convenient tool to help us to distinguish crucial metastasis genes and related pathway from other ambiguous, redundant and broad genes network that is valuable for us to interpret the intricate EMT mechanism.

Citation Format: Xiaoping Zhu, Yin Zhen, Hong Zhao, Stephen Wong. High-content imaging facilities modeling of cancer epithelial-mesenchymal transition signaling network. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C2.