bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B16: Extracellular S100A4 as an inducer of a cytokine network that mediates tumor-stroma crosstalk: Potential implications for metastases
Authors: Bettum Ingrid J. , Vasiliauskaite Kotryna , Pettersen Solveig J. , Malandsmo Gunhild M. , Prasmickaite Lina
Published: August 2014

Among soluble factors that appear to be of high importance for metastasis progression are chemokines from the S100 family. One such factor is S100A4, a small calcium binding protein linked to metastasis and poor prognosis in several cancer types. The protein is expressed in and secreted from not only tumor cells, but also various stroma cells, suggesting that S100A4 might be an important factor in the tumor microenvironment. Recently we have revealed that in vivo S100A4-positive stroma cells are tightly associated with the metastatic nodules in the brain and lungs, suggesting the role of S100A4 in facilitating metastatic growth in these microenvironments. How S100A4 executes its metastasis-promoting functions is however not fully elucidated. Here we hypothesize that the extracellular S100A4 can trigger a cytokine network that mediates the tumor-stroma crosstalk and thereby facilitates metastases progression in malignant melanoma. We have shown that extracellular S100A4 stimulates melanoma cells to secrete various cytokines and growth factors, including IL-8, IL-6, CXCL2, sICAM-1 and VEGF, molecules playing a role in cancer progression and angiogenesis in particular. Since cytokines can act in a paracrine manner, they can mediate interactions between tumor cells and stroma cells. Thus, we have shown that conditioned medium from melanoma cells treated with extracellular S100A4 modulates the angiogenic functions of endothelial cells making them more migratory and more proned to make vascular-like networks in vitro. Among factors induced by S100A4 were also pro-inflammatory cytokines associated with macrophage recruitment and/or activation. Currently we are investigating whether the S100A4-triggered cytokines modulate the properties of macrophages making them support tumor cells. Altogether, our results indicate a potential role of S100A4 in stimulating tumor-stoma crosstalk which might mimic the formation of a metastatic niche-like milieu facilitating metastases.

Citation Format: Ingrid J. Bettum, Kotryna Vasiliauskaite, Solveig J. Pettersen, Gunhild M. Malandsmo, Lina Prasmickaite. Extracellular S100A4 as an inducer of a cytokine network that mediates tumor-stroma crosstalk: Potential implications for metastases. [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 B16.
Abstract B17: A microscale 3-D in vitro model of the breast cancer progression from DCIS to IDC: Deciphering the role of the stromal fibroblasts
Authors: Sung Kyung Eun , Pehlke Carolyn , Berthier Erwin , Eliceiri Kevin W. , Friedl Andreas , Beebe David J.
Published: August 2014

Breast cancer progression from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) is a critical step in breast cancer. This invasive transition of DCIS is defined by stromal invasion and is a life-threatening step accompanied by a dramatic drop in prognosis. In addition, the invasive transition of DCIS is largely driven by stromal alteration; thus it is critical to understand how DCIS alters the surrounding microenvironment, thus causing the cancer cells to become invasive. This study investigates the role of stromal fibroblasts in the vicinity of DCIS during the invasive transition using an innovative multidisciplinary approach that combines 3D cancer biology, microfluidics, and high-resolution imaging. We recently developed an efficient 3D microfluidic system that supports the transition from DCIS to IDC. The in vitro system employs microchannels with two inputs and one output enabling MCF10-DCIS.com cells (MCF-DCIS) and human mammary fibroblasts (HMF) to be loaded in two adjacent (side-by-side) compartments. This platform allows investigations of effects of spatial organization on the transition by independently analyzing their morphology and the modifications to the surrounding collagen architecture. Importantly, the compartmentalized platform enables monitoring of both MCF-DCIS and HMF independently including quantitative measures of the collagen architecture associated with each cell type. We observed that the HMF near MCF-DCIS became more protrusive versus HMF relatively far from MCF-DCIS. We have also begun to identify how the HMF become activated and protrusive when co-cultured with MCF-DCIS and to understand the biological function and impact of protrusive HMF during DCIS progression to IDC. We verified that the signaling based on Cathepsin D produced from MCF-DCIS and low-density lipoprotein receptor-related protein-1 (LRP1) from HMF is involved in regulation of the protrusive activity of HMF. Additionally, knocking down LRP1 in HMF inhibited the invasive transition of MCF-DCIS. This study demonstrates one possible route through which MCF-DCIS activate pre-existing fibroblasts and subsequently, leads to the modification of the ECM and the progression to IDC.

Citation Format: Kyung Eun Sung, Carolyn Pehlke, Erwin Berthier, Kevin W. Eliceiri, Andreas Friedl, David J. Beebe. A microscale 3-D in vitro model of the breast cancer progression from DCIS to IDC: Deciphering the role of the stromal fibroblasts. [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 B17.
Abstract B23: Tumor-associated macrophages in human papillary thyroid cancer
Authors: Cho Sun Wook , Min Hye Sook , Kim Seunwhan , Kim Kyung Won , Park Do Joon , Park Young Joo
Published: August 2014

Introduction: Tumor associated macrophages (TAMs) play a pivotal role in tumoriogenesis of human cancers, including advanced thyroid cancers. A recent study demonstrated that the increased density of TAMs was positively associated with lymph node (LN) metastasis in papillary thyroid cancer (PTC). The purpose of this study was to investigate the functional role of TMA in PTC. Method: Immunohistochemistry with anti-CD68 antibody was performed in 35 PTC patients who had LN metastasis. Co-culture of human PTC (SNU-790) cells and activated human monocytes (THP-1) by macrophage-colony stimulating factor (M-CSF, 50ng/ml) were performed and cell survival rates were analyzed by MTT assay. Result: Among 35 patients, 9 were male and 26 were females. Mean age was 48.9 ± 13.4 years and mean tumor size was 0.94 ± 0.51 cm. Twenty six (74.2%) patients had extrathyroidal invasion. Immunohistochemical staining of primary tumor showed that the expression of CD68 was presence in 3~15% of the peri- and intratumoral areas in all cases, while there was no positivity in normal thyroid tissue adjacent to tumors. In vitro stimulation of THP-1 cells with M-CSF showed significant increase of IL-10 by 7~8 fold compared to vehicle treated cells. Co-culture of SNU-790 cells with M-CSF stimulated THP-1 cells showed higher survival rates than control. Conclusions: TAMs exist in invasive human PTC specifically in intratumoral areas. Activated human monocytes, representing TAM phenotypes support tumor cell survival. Collectively, TAMs may have a functional role in tumor microenvironment of human PTC.

Citation Format: Sun Wook Cho, Hye Sook Min, Seunwhan Kim, Kyung Won Kim, Do Joon Park, Young Joo Park. Tumor-associated macrophages in human papillary thyroid 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 B23.
Abstract B28: Signaling by PDGF-CC in the maintenance of a protumorigenic breast cancer microenvironment
Authors: Roswall Pernilla , Haller Kristian , Petersson Annika , Anderberg Charlotte , Li Hong , Norum Jens-Henrik , Toftgård Rune , Eriksson Ulf , Pietras Kristian
Published: August 2014

It is increasingly appreciated that cancer results from the concerted performance of genetically altered tumor cells interacting with ostensibly normal cells in the microenvironment. As the cancer progresses, the surrounding stroma co-evolves into an activated state through continuous paracrine communication, thus creating a dynamic signaling circuitry that promotes cancer initiation and growth, and ultimately leads to a fatal disease. We aim to molecularly map the support functions performed by the various cell types comprising the tumor stroma. Specifically we will focus on, cancer-associated fibroblasts (CAFs), which are known to support many aspects of tumor progression. Based on our previous finding that paracrine signaling by Platelet-derived growth factor (PDGF)-CC serves to recruit CAFs into tumors we have generated Pdgfc-deficient genetically engineered mouse models of breast cancer. Our results show a significant reduction in the tumor burden followed by prolonged survival in the absence of PDGF-CC, in agreement with the recent finding that PDGF-CC expression is an indicator of poor prognosis for breast cancer patients. Upon transplantation of small tumor pieces from wildtype and Pdgfc-deficient tumors respectively into the mammary fatpad of wildtype mice, Pdgfc-deficient tumors show substantial growth retardation with massive hemorrhage and necrosis, indicative of malfunctional vessels and insufficient blood supply. qPCR arrays on total RNA from wildtype and Pdgfc-deficient tumors respectively, suggest that lack of PDGF-CC causes perturbed differentiation yielding a less advanced tumor. Furthermore, we are currently investigating the differences observed in the recruited fibroblasts and/or MSCs in wildtype and Pdgfc-deficient tumors, by performing proteomics on isolated PDGFR-α positive cells. Tumor morphology in combination with proteomics suggests that the lack of PDGF-CC causes an impaired migration and/or guidance of the recruited fibroblasts from the periphery of the tumor. Thus, our studies suggest a novel role for PDGF-CC in creating a pro-tumorigenic microenvironment and identify PDGF-CC as a valid and promising therapeutic target in breast cancer.

Citation Format: Pernilla Roswall, Kristian Haller, Annika Petersson, Charlotte Anderberg, Hong Li, Jens-Henrik Norum, Rune Toftgård, Ulf Eriksson, Kristian Pietras. Signaling by PDGF-CC in the maintenance of a protumorigenic breast cancer microenvironment. [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 B28.
Abstract B35: The effect of extracellular matrix proteins from lung on the migration of breast cancer cells
Authors: Carpenter Philip M. , Xiao Cally , Ngo Tuan , Gershon Paul D.
Published: August 2014

The lung is a frequent site of breast carcinoma metastasis, yet little is known about the mechanism of breast carcinoma metastasis to lung. The purpose of this study was to determine if extracellular matrix components from the pulmonary microenvironment had the potential to encourage metastasis by inducing motility in breast carcinoma cells. Previous studies provided evidence that products of conditioned media of pulmonary epithelial cell co-cultures were able to induce dose-dependent motility in breast carcinoma cells. The deposited material was analyzed by mass spectrometry, revealing extracellular matrix proteins, including thrombospondin 1 (TSP), tenascin C, transforming growth factor-beta-induced protein ig-h3 (βig-h3), and the α3 and β3 chains of laminin 332 (LN332). Motility assays using either recombinant preparations of TSP, βig-h3 or tenascin C, or purified LN332 revealed that LN332 induced greater motility at almost all concentrations of each protein tested.

Since pulmonary cells grown on membranes at a liquid air interface more closely resemble the structure of lung tissue in vivo, cells grown in this manner were tested for their ability to produce LN332 and induce motility. After 7-8 days in culture, pulmonary epithelial cells provided a monolayer at the liquid air interface. Membranes with cells on the surface exposed to air were fixed in formalin, paraffin embedded and sectioned for hematoxylin and eosin staining and immunohistochemistry. Immunohistochemistry revealed that the cells produced LN332, which was both cytoplasmic and in a layer on the membrane surface beneath the cells. Lung cells growing at the liquid air interface were removed from the membrane, and MCF-7 cells were added to the same membrane from which lung cells had been removed, or to membranes which had been incubated with pulmonary cell growth media but no lung cells. Whereas MCF-7 cells placed on membranes without prior lung cells grew in tight clusters, MCF-7 cells added to membranes on which lung cells had been removed showed scattering and pseudopodia formation. This response was blocked by antibodies to α3 and β1 integrins, which are receptors of LN332. Finally, western blots of human lung tissue revealed the presence of LN332. These experiments together showed that pulmonary LN332 is a likely motility factor for breast carcinoma cells. These findings provide a mechanism for the migration of metastatic tumor cells from their arrest in the pulmonary vasculature into the surrounding tissue, allowing their establishment as a metastatic colony.

Citation Format: Philip M. Carpenter, Cally Xiao, Tuan Ngo, Paul D. Gershon. The effect of extracellular matrix proteins from lung on the migration of breast 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 B35.
Abstract B36: Understanding the mechanisms underlying recurrence of malignant gliomas after antiangiogenesis treatment
Authors: Santiago Nahir Cortes , Gabrusiewicz Konrad , Liu Dan , Hossain Mohammed B. , Conrad Charles , Fueyo Juan , Gomez-Manzano Candelaria
Published: August 2014

Anti-angiogenesis therapies, in particular bevacizumab, are currently offered to patients with malignant gliomas given strong preclinical and clinical data supporting its effectiveness. However, evidence gathered in the literature shows that upon bevacizumab treatment patients will invariably recur with highly invasive tumors resistant to all available therapies. The purpose of the study we have undertaken is to understand the underlying mechanisms involved in this enhanced malignancy with the aim to uncover new combined therapeutic strategies for these patients. Using tissue obtained from glioma bearing mice treated with bevacizumab or control treatment, we have obtained evidence that upon anti-VEGF treatment, a population of Tie2-expressing cells is recruited to the tumor particularly around areas of invasion. Further characterization of this population demonstrated that a significant proportion of these cells are of myeloid origin, Tie2-expressing monocytes or TEMs. Through the use of an M2 polarized culture as well as TEMs obtained from blood of healthy donors we have gathered data supporting that this myeloid cell population has high expression of several transcripts of molecules associated with invasion as well as a much higher secretion of MMP2 and MMP9 than their Tie2 negative counterparts. In an attempt to understand the changes within the tumor microenvironment after anti-VEGF therapy, immunohistochemical analysis for expression of several molecules associated with angiogenesis was performed. Results obtained identified angiopoietin 2 (Ang2), a Tie2 ligand, as being dramatically increased after therapy. Interestingly, our in vitro and in vivo data show that Ang2 serves as a chemo-attractant molecule to TEMs and therefore could be the main molecule responsible for their recruitment into the tumors following anti-VEGF treatment. We gathered preliminary data on the enhanced presence of TEMs as well as higher levels of MMP9 in human surgical samples after bevacizumab treatment. Our data strongly supports a strong influence of TEMs and Ang2 in the recurrent phenotype of gliomas after anti-VEGF therapy. Further studies aim to understand the extent to which targeting Ang2 specifically and the Ang/Tie2 pathway in general, in combination with anti-VEGF therapy, will lead to improved outcomes.

Citation Format: Nahir Cortes Santiago, Konrad Gabrusiewicz, Dan Liu, Mohammed B. Hossain, Charles Conrad, Juan Fueyo, Candelaria Gomez-Manzano. Understanding the mechanisms underlying recurrence of malignant gliomas after antiangiogenesis treatment. [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 B36.
Abstract B38: Unveiling the mechanisms of melanoma metastasis using a chick embryo neural crest model system
Authors: Bailey Caleb M. , Kulesa Paul M.
Published: August 2014

In an attempt to elucidate the mechanisms of melanoma metastasis, our laboratory has recently performed a molecular comparison between melanoma and the embryonic neural crest. The neural crest is a multi-potent and highly invasive cell population, and represents the ancestral cell type of melanocytes. We report that melanoma cells exploit portions of the embryonic neural crest development program to facilitate invasion. In particular, Eph receptor tyrosine kinases and their cognate ligands, the ephrins, are dynamically regulated by C8161 melanoma cells to facilitate migration. We noted that the purported metastasis suppressor EphB6 was significantly reduced in invasive melanoma cells compared to poorly invasive melanoma cells and primary human melanocytes. In the current study, we have utilized the chick embryo transplant model system to examine the effects of EphB6 re-expression in C8161 melanoma cells. EphB6 over-expression significantly alters cell directionality of migrating melanoma cells within the embryo. We confirmed the anti-metastatic effect of EphB6 in melanoma cells using a chick CAM metastasis assay, which demonstrated a significant decrease in metastatic potential. Importantly, tumor formation appeared unaffected. These results suggest that EphB6 acts specifically as a metastasis suppressor, likely at the metastatic step of intravasation. Our studies also demonstrate the chick embryo as a powerful tool with which to compare and dissect the complex mechanisms of metastasis in vivo that are similar or distinct from normal developmental processes.

Citation Format: Caleb M. Bailey, Paul M. Kulesa. Unveiling the mechanisms of melanoma metastasis using a chick embryo neural crest model system. [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 B38.
Abstract B39: Inhibiting endothelial FAK activity blocks tumor cell extravasation
Authors: Jean Chris , Nam Ju-Ock , Chen Xiao-Lei , Uryu Sean , Tancioni Isabelle , Lawson Christine , Ward Kristy K. , Walsh Colin T. , Ghassemian Majid , Turowski Patric , Dejana Elisabetta , Weis Sara M. , Cheresh David A. , Schlaepfer David D.
Published: August 2014

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that modulates signaling from growth factor, integrin, and cadherin receptors in both tumor and stromal cells. Pharmacological FAK inhibition prevents tumor growth and metastasis, but the target cell action associated with this remains unclear. Recent studies using an inducible endothelial cell (EC) specific FAK kinase-inactive knock-in mouse model revealed that vascular endothelial growth factor (VEGF) increased cell permeability was dependent upon FAK activity. VEGF triggers conformational FAK activation, binding of FAK to the cytoplasmic tail of VE-cadherin, and FAK-dependent phosphorylation of adherens junction proteins regulating vascular integrity. Here we provide genetic and pharmacological support for the importance for FAK activity in mediating direct VE-cadherin tyrosine (Y) 658 phosphorylation in tumor-associated ECs in vivo. In cell culture, VEGF-induced transmigration of tumor cells through an EC barrier is prevented by genetic EC-specific FAK inhibition or reconstitution of Y658F VE-cadherin within null ECs. In mice, VEGF enhances circulating tumor cell extravasation within the lungs and this is prevented by inducible knockin of kinase-dead FAK within ECs. These studies provide a mechanistic foundation for the importance of FAK and VE-cadherin interactions in the regulation of adherens junction stability and support future evaluation of FAK inhibitors in the treatment of metastatic cancer.

Citation Format: Chris Jean, Ju-Ock Nam, Xiao-Lei Chen, Sean Uryu, Isabelle Tancioni, Christine Lawson, Kristy K. Ward, Colin T. Walsh, Majid Ghassemian, Patric Turowski, Elisabetta Dejana, Sara M. Weis, David A. Cheresh, David D. Schlaepfer. Inhibiting endothelial FAK activity blocks tumor cell extravasation. [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 B39.
Abstract B4: Development of a short peptide with antitumor activity
Authors: Wang Suming , Catena Raul , Mittal Vivek , Watnick Randolph S.
Published: August 2014

It has been demonstrated that metastatic tumors establish bone marrow (BM)-derived pre-metastatic niches in distant organs that serve as permissive hubs for supporting future metastases. It is not known, conversely, whether tumors that lack metastatic potential fail to establish such niches, or generate niches which inhibit metastasis. Here, we show that metastasis-incompetent tumors are also capable of generating BM-derived niches at distant sites. However, by secreting prosaposin (psap) these tumors systemically stimulate expression of an antiangiogenic factor thrombospondin1 (Tsp-1) in myeloid progenitor cells in the niche, converting them into metastasis-inhibitory cells. We found that Tsp-1-inducing activity of psap was contained in a short (<10 amino acid) peptide. Importantly this peptide suppressed metastatic outgrowth, which was rescued by genetic ablation of Tsp-1 in the BM. These results provide mechanistic insights into why certain tumors are deficient in metastatic potential, and unexpectedly implicate recruited Gr1+ myeloid progenitor cells as the main source of Tsp-1 in the metastatic lung. Clinically, psap expression was significantly decreased in skeletal metastases in prostate cancer patients, suggesting that the psap peptide may be a potential therapeutic agent against metastatic cancer.

Citation Format: Suming Wang, Raul Catena, Vivek Mittal, Randolph S. Watnick. Development of a short peptide with antitumor activity. [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 B4.
Abstract B43: A novel breast cancer metastasis suppressor upstream of Hippo signaling
Authors: Ma Li
Published: August 2014

There is a pressing need to identify prognostic markers of metastatic diseases and targets for treatment. Combining high-throughput RNA sequencing, functional characterization, mechanistic studies, and clinical validation, we identify leukemia inhibitory factor receptor (LIFR) as a breast cancer metastasis suppressor downstream of the miR-9 microRNA and upstream of Hippo signaling. Restoring LIFR expression in highly malignant tumor cells suppresses metastasis by triggering a Hippo kinase cascade that leads to phosphorylation, cytoplasmic retention, and functional inactivation of the transcriptional co-activator YAP. Conversely, loss of LIFR in non-metastatic breast cancer cells induces migration, invasion, and metastatic colonization through activation of YAP. LIFR is downregulated in human breast carcinomas and inversely correlates with metastasis. Notably, loss of LIFR in non-metastatic Stage I-III breast tumors is highly associated with poor metastasis-free, recurrence-free and overall survival outcomes in approximately 1,000 patients. These findings identify LIFR as a metastasis suppressor that functions through the Hippo-YAP pathway and has significant prognostic power. As the next step, we will dissect the role of LIFR in spontaneous breast tumorigenesis and metastasis using genetically engineered mouse models, elucidate the mechanisms by which LIFR activates Hippo signaling using biochemical approaches, and develop methods to restore LIFR expression or function as new anti-metastatic therapeutic strategies.

Citation Format: Li Ma. A novel breast cancer metastasis suppressor upstream of Hippo signaling. [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 B43.
Abstract B54: Coordinate RB/PTEN tumor suppressor loss: Drives aggressive invasive behavior and is strongly associated with the progression of ductal carcinoma to invasive breast cancer
Authors: Witkiewcz Agnieszka , Pajak Thomas , Schwartz Gordon , Knudsen Erik
Published: August 2014

Background: A subset of patients with ductal carcinoma in situ (DCIS) treated by surgery will progress to invasive breast cancer. However, there is a limited understanding of the pathways driving such invasive progression and no accepted markers to differentiate lesions at high risk from those at lower risk of developing invasive disease.

Methods: The association of two tumor suppressor genes, retinoblastoma (RB) and phosphatase and tensin homolog (PTEN), with risk of any ipsilateral breast event (IBE) or progression to invasive breast cancer (IBC) was analyzed using data from 236 DCIS patients treated with breast conserving surgery with long-term follow up. RB and PTEN expression was assessed with immunohistochemistry. The functional effects of RB and/or PTEN loss were evaluated in preclinical models.

Results: Modeling of RB and PTEN loss as observed in DCIS, revealed that the tumor suppressors coordinate complementary processes with respect to proliferation, motility, and invasiveness. Correspondingly, pharmaceutical interventions that mimic activation of RB or PTEN limit the invasive properties of such models. Gene expression profiling demonstrated unique transcriptional programs regulated by each tumor suppressor that are associated with epithelial mesenchymal transition and invasive disease progression. Loss of RB immunoreactivity in DCIS was strongly associated with risk of IBE occurrence (HR=2.64, 95% CI = 1.64 to 4.25) and IBC recurrence (HR=4.66, 95% CI = 2.19 to 9.93). The prognostic power of RB loss alone remained statistically significant in multivariable analyses, although PTEN loss alone did not. However, patients with DCIS lesions that were both RB and PTEN deficient were at higher risk for IBE (HR=3.39, 95% CI = 1.92 to 5.99) and IBC recurrence (HR=6.1, 95% CI = 2.5 to 14.76).

Conclusions: These studies indicate that RB and PTEN have independent functions that modulate the invasive potetntial of breast cancer, and together have prognostic utility that could be used to improve the management of DCIS.

Citation Format: Agnieszka Witkiewcz, Thomas Pajak, Gordon Schwartz, Erik Knudsen. Coordinate RB/PTEN tumor suppressor loss: Drives aggressive invasive behavior and is strongly associated with the progression of ductal carcinoma to invasive breast 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 B54.
Abstract B59: The dual effect of therapy-induced IL-1β expression on tumor progression: Role of tumor-associated macrophages
Authors: Voloshin Tali , Alishekevitz Dror , Miller Valeria , Isakov Lika , Bril Rotem , Benhar Moran , Apte Roney , Shaked Yuval
Published: August 2014

A major obstacle in clinical oncology is that tumors eventually acquire resistance to therapy. The mechanism by which resistance arises is usually associated with changes made in tumor cells in response to the therapy, however, little is known about the effect of the host which may also contribute to tumor resistance. Previous studies from our lab suggest that the induction of host cellular and molecular events following therapy may promote tumor re-growth, angiogenesis and metastasis. In this study we uncovered the possible role IL-1β - a proinflammatory cytokine - has on the growth of primary tumor and its impact on metastasis following paclitaxel (PTX) chemotherapy. We found that plasma and spleen from PTX-treated mice exhibited elevated expression of IL-1β. Furthermore, plasma from PTX-treated mice induced tumor cell invasion, an effect which was abrogated in plasma from mice treated with PTX and Anakinra, an IL-1 receptor antagonist. Consequently, mice inoculated with Lewis lung carcinoma (LLC) cells pre-exposed to plasma from mice treated with the combined therapy survived longer than mice inoculated with tumor cells pre-exposed to plasma from mice treated with PTX or Anakinra monotherapy. We next determined whether treatment efficacy of PTX is enhanced by the combination of PTX and Anakinra using 4T1 metastatic breast cancer model in mice. Tumors treated with the combined therapy were significantly smaller than tumors treated with PTX or Anakinra monotherapy. Surprisingly, although the primary tumors were smaller, such mice succumb to metastasis earlier than mice in any of the other treated groups. Furthermore, an increased number of M2 macrophages colonizing 4T1 primary tumors treated with the combined therapy was observed when compared to their number in PTX treated tumors, indicating a possible pro-metastatic role of M2 macrophages in post treatment IL-1β deprived microenvironment. Parallel results were observed when LLC bearing IL-1β deficient mice, were treated with PTX. Taken together, these findings demonstrate that although treatment outcome is enhanced following the blockade of host-induced IL1β at the primary tumor level, at the metastatic level it may serve as a double-edge sward and can lead to metastatic spread.

Citation Format: Tali Voloshin, Dror Alishekevitz, Valeria Miller, Lika Isakov, Rotem Bril, Moran Benhar, Roney Apte, Yuval Shaked. The dual effect of therapy-induced IL-1β expression on tumor progression: Role of tumor-associated macrophages. [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 B59.
Abstract B63: Cancer cell invasion determined by cell density-dependent, cytokine-mediated crosstalk
Authors: Sharif Ghada M. , Schmidt Marcel O. , Hu Zhang-Zhi , Glasgow Eric , Riegel Anna T. , Wellstein Anton
Published: August 2014

Metastatic spread of cancer cells from their primary site requires invasion into the vasculature, evasion at the distant organ site and colonization of the distant organ. Here we studied mechanisms of attachment and invasion into endothelial monolayers by cancer cells to identify driver molecules and signaling pathways that are crucial for the invasive and metastatic phenotype. In vitro studies were complemented by studies in zebrafish embryos and mouse models of cancer cell invasion and metastasis. In one approach we compared gene expression profiles across isogenic cell lines with distinct invasive behavior to identify candidate drivers and pathways. These were then subjected to functional validation. This comparison of gene expression profiles of isogenic cancer cell lines showed an upregulation of immune response cytokines associated with an endothelial invasive phenotype in human breast cancer cells. The role of the respective cytokine pathways was validated by blocking the respective receptors in in vitro assays and in a zebrafish metastasis model.

Citation Format: Ghada M. Sharif, Marcel O. Schmidt, Zhang-Zhi Hu, Eric Glasgow, Anna T. Riegel, Anton Wellstein. Cancer cell invasion determined by cell density-dependent, cytokine-mediated crosstalk. [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 B63.
Abstract B71: Syndecan-1 promotes breast cancer metastasis by modulating tissue stiffness
Authors: Chute Colleen L. , Keely Patricia , Alexander Caroline , Friedl Andreas
Published: August 2014

Tumor-stroma interactions are integral to breast cancer progression and metastasis. It has been well established that women with dense breast tissue have increased risk of developing breast cancer as compared to women with less dense breast tissue; it has also been documented that a stiffer extracellular matrix (ECM) can promote tumor cell invasion both in vitro and in vivo. Our lab has previously shown that Syndecan-1 (Sdc1), a cell surface heparan sulfate proteoglycan, regulates the in vitro organization of a fibroblast-constructed ECM that thus creates an invasion-permissive microenvironment. Therefore, we sought to characterize the role of stromal Sdc1 in cancer progression in vivo. Using the 4T1 model of late stage metastatic breast cancer in both Sdc1 wild-type (WT) and Sdc1 knock-out (KO) mice, we assayed for tumor metastasis from orthotopic tumor cell implants. Sdc1 deficiency in the host did not influence the primary tumor weight or growth rate, but significantly reduced the incidence and the number of metastatic lesions (p<0.001, p<0.0001, respectively). In an effort to dissect out the mechanism by which loss of stromal Sdc1 is inhibiting metastasis, we analyzed the tumor microenvironment for the presence of specific cell types and structural differences. There was no difference in the recruitment of macrophages, the activation of fibroblasts, angiogenesis, or the structure or deposition of collagen between 4T1-induced tumors in the Sdc1 WT and Sdc1 KO mice. Interestingly, preliminary data suggest the tumors from the Sdc1 WT mice are significantly stiffer than the tumors from Sdc1 KO mice, as measured by atomic force microscopy. To determine whether Sdc1 was influencing later stages of metastasis we injected tumor cells via tail vein to specifically assay extravasation and metastatic outgrowth. After 2 weeks, all mice assayed had metastatic lesions but the Sdc1 WT mice had significantly more numerous and larger lesions (p<0.004, p<0.05, respectively). These data indicate that the presence of stromal Sdc1 promotes tumor cell outgrowth in the metastatic target organ, but may not play as large a role in tumor cell extravasation based on the finding that all mice assayed had metastatic lesions. These results further suggest that Sdc1 influences breast cancer metastasis by promoting increased tissue stiffness, which promotes tumor cell invasion and tumor outgrowth at secondary metastatic target organs, and ultimately promotes formation of metastatic lesions.

Citation Format: Colleen L. Chute, Patricia Keely, Caroline Alexander, Andreas Friedl. Syndecan-1 promotes breast cancer metastasis by modulating tissue stiffness. [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 B71.
Abstract B73: Unraveling the role of a novel endoplasmic reticulum protein in cancer cell migration
Authors: Evensen Nikki Ann , Kuscu Cem , Dufour Antoine , Zucker Stanley , Cao Jian
Published: August 2014

Although considerable progress has been made in our understanding of cancer cell invasion, much of this complex cellular process remains a mystery, ultimately hindering advancements in treatment strategies targeting metastasis. Here we report, for the first time, the vital role of a novel endoplasmic reticulum (ER) resident protein identified by a PCR-subtraction hybridization technique, designated Cancer ER Invasion Gene (CERIG), in cancer cell migration and invasion. Clinical relevance of CERIG is highlighted by upregulation of CERIG in human invasive breast cancer specimens examined by immunohistochemistry and real-time RT-PCR, as well as increased expression in various other forms of human cancer as determined by data mining. This up-regulation of CERIG correlates with poor prognosis of patients with breast cancer assessed by a DNA microarray data-mining approach from three publicly available cohorts containing a total of 696 breast cancer patients. Silencing of CERIG in metastatic MDA-MB-435 cancer cells disrupted mesenchymal morphogenesis in vitro, resulting in a mesenchymal-to-epithelial transition, and interfered with cancer metastasis in vivo. Employing both gain- and loss-of-function approaches, CERIG was found to induce cancer cell migration and invasion. Enhanced cell migration required retention of CERIG within the ER, where this protein forms a stable complex with the ER chaperone protein, GRP78/BiP. Mutagenesis studies identified a novel ER retention motif within CERIG required for its ER localization, BiP interaction, and enhanced migration. Furthermore, fusion of the ER-retention domain to a soluble matrix metalloproteinase (MMP) prevents the secretion of the MMP by retaining it in the ER. Mechanistically, overexpression of CERIG results in ER calcium leakage via the inositol trisphosphate receptor (IP3R) and a subsequent increase in cytosolic calcium, ultimately resulting in activation of PKCα and cell migration. Moreover, chelating intracellular calcium abrogates CERIG induced PKCα activation and cell migration. Taken together, our data indicate that CERIG, as a novel ER resident protein, plays an important role in cancer progression, serving as a novel cell migration-promoting gene.

Citation Format: Nikki Ann Evensen, Cem Kuscu, Antoine Dufour, Stanley Zucker, Jian Cao. Unraveling the role of a novel endoplasmic reticulum protein in cancer cell migration. [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 B73.
Abstract B77: Curcumin deteriorates trabecular and cortical bone in mice bearing metastatic Lewis lung carcinoma
Authors: Yan Lin
Published: August 2014

Bone is a major target of metastasis for many malignancies; curcumin has been studied for its role in cancer prevention including early phase clinical trials for its efficacy and safe use with cancer patients. The present study investigated the effects of dietary supplementation with curcumin (2% and 4%) on (1) pulmonary metastasis of Lewis lung carcinoma and (2) bone microstructural changes in female C57BL/6 mice. Curcumin significantly increased the size of lung metastases, which was accompanied with significant increases in plasma concentrations of angiogenic factors (e.g. angiogenin, VEGF) and inflammatory cytokines (e.g. IL-1β, MCP-1). Morphometric analysis showed that curcumin at 2% and 4% dietary levels significantly reduced bone volume to total volume ratio (BV/TV), connective density (Conn.D.) and trabecular number (Tb.N.) and significantly increased structure model index (SMI, an indicator of the plate- and rod-like geometry of trabecular structure) and trabecular separation (Tb.Sp.) in vertebral bodies, and at the 4% level curcumin significantly reduced BV/TV and Tb.N. and at either level it significantly reduced trabecular thickness (Tb.Th.) in distal femurs compared with the controls. In the mid-shaft of femur, curcumin at the 4% level significantly reduced cortical bone area to total area ratio (Ct.Ar/Tt.Ar.) and cortical thickness (Ct.Th.) compared with the controls. Furthermore, curcumin feeding resulted in dose-dependent decreases in plasma concentrations of osteocalcin (a marker of bone formation) and increases plasma tartrate-resistant acid phosphate 5b (a marker of bone resorption) compared with the controls, indicating that curcumin may deteriorate trabecular and cortical bone by disrupting the balance of bone remodeling. As skeleton is a favored site of metastasis for many cancers, our results that curcumin (1) enhances metastatic growth and (2) results in osteolytic deterioration of bone suggest the need of assessment of bone structural changes for curcumin cancer prevention studies, particularly for human trials with cancer patients who are the risk of malignant spread, to determine the curcumin effects and its interaction with malignancy on skeleton health.

Citation Format: Lin Yan. Curcumin deteriorates trabecular and cortical bone in mice bearing metastatic Lewis lung 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 B77.
Abstract B81: Angiomodulin (AGM/IGFBP-rP1) is overexpressed in tumor vasculature and regulates adhesion of vascular endothelial cells via integrin αvβ3: Possible roles in tumor angiogenesis
Authors: Miyazaki Kaoru , Komiya Eriko , Sato Hiroki , Miyagi Yohei , Higashi Shouichi
Published: August 2014

Angiomodulin (AGM) is a member of insulin-like growth factor binding protein (IGFBP) superfamily and often called IGFBP-rP1. Our past studies identified this protein as a tumor-derived cell adhesion factor (TAF) that is highly accumulated in blood vessels of human cancer tissues (PNAS 93:8384, 1996). Recently we found that AGM is overexpressed in not only vasculature but also stromal fibroblasts in some human cancers (Cancer Sci 4:691, 2012). AGM stimulates the growth of fibroblasts and their expression of fibronectin and α-smooth muscle actin, inducing their morphological change to a myofibroblast-like shape. It is supposed that AGM activates cancer-associated fibroblasts (CAFs) and increases cancer stroma. In contrast to these findings, tumor-suppressing activity of AGM has been reported by other studies. To clarify the roles of AGM in tumor progression, we here investigated the distribution of AGM in benign and invasive breast cancers and its regulatory factors and biological activities. Immunohistochemical analysis showed that AGM was greatly expressed in tumor vasculature even in ductal carcinoma in situ (DCIS) as compared to normal vasculature, while its expression in CAFs was more prominent in invasive carcinomas than DCIS. In vitro analyses showed that AGM was markedly induced by VEGF in vascular endothelial cells but by TGF-β in fibroblasts. Although AGM did not significantly affect the growth of endothelial cells, it potently promoted the adhesion of endothelial cells through integrins. Experiments with neutral anti-integrin antibodies revealed that the cell adhesion activity was mediated mainly by integrin αvβ3 and weakly by integrin α2β1. AGM induced cytoskeletal change in endothelial cells. Furthermore, AGM and integrin αvβ3 were colocalized at high levels in tumor vasculature. These results suggest that AGM regulates angiogenesis and other vascular functions as a ligand of integrin αvβ3 in cancer tissues. The data also indicate that AGM is induced by different mechanisms and plays different roles in tumor progression between CAFs and vasculature.

Citation Format: Kaoru Miyazaki, Eriko Komiya, Hiroki Sato, Yohei Miyagi, Shouichi Higashi. Angiomodulin (AGM/IGFBP-rP1) is overexpressed in tumor vasculature and regulates adhesion of vascular endothelial cells via integrin αvβ3: Possible roles in tumor angiogenesis. [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 B81.
Abstract B87: Antiangiogenic and antimetastatic activities of cat's whiskers tea (Orthosiphon stamineus) extract against colon cancer cell line
Authors: Alsuede Fouad Saleih , Majid Aman Shah Abdul , B Khadeer Ahamed Mohamed , Ismail Z , Kadir M.O , Majid Amin Malik Shah Abdul
Published: August 2014

Orthosiphon stamineus is a popular medicinal herbal tea that is traditionally used for inflammatory joint condition, diabetes and hypertension. In this study we found that the 50% ethanolic extract of O. stamineus strongly suppressed angiogenesis in isolated rat aortic tissues and prevented the formation of chorionic plexus in chick embryo. The extract also inhibited the formation of blood vessels in matrigel plug implanted in mice. A significant inhibition of important stages of HCT-116 colon cancer cells metastasis, invasion, and migration was also observed with significant inhibitory effect of on the growth of the neoplastic cells. However, the extract displayed low cytotoxicity towards human tumor cell lines such as K562, HL-60, Skno-1, MCF-7, HCT-116 and Hep G2, and normal human colonic fibroblast CCD-18Co. HPLC analysis reveals that the extract is abundant with rosmarinic acid, eupatorin, sinensetin, etc. This work highlights usefulness of O. stamineus in treating colon cancer.

Note: This abstract was not presented at the conference.

Citation Format: Fouad Saleih Alsuede, Aman Shah Abdul Majid, Khadeer Ahamed Mohamed B, Z Ismail, M.O Kadir, Amin Malik Shah Abdul Majid. Antiangiogenic and antimetastatic activities of cat's whiskers tea (Orthosiphon stamineus) extract against colon cancer cell line. [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 B87.
Abstract C13: The role of cancer heterogeneity in metastasis
Authors: Shin Yoojin , Jeong Hyo Eun , Han Sewoon , Won Jihee , Chung Seok
Published: August 2014

Breast cancer doesn't contain only one colony of tumor cells. It appears to have heterogeneous cell composition of characteristically different subtypes with distinct molecular and gene expression signatures. Due to the tumor heterogeneity, which determines the risk of metastatic progression and drug resistance, it is difficult to predict progression patterns of cancer and provide the most suitable treatment to patients.

Most research has focused on molecular definition and genetic association for the cancer heterogeneity studies. However, lack of in vitro models to precisely mimic the heterogeneous conditions of tumors has made it difficult to understand the role of heterogeneity in metastasis, especially invasion.

So, to precisely regulate heterogeneous microenvironment of tumors and evaluate the role of the heterogeneity on invasion & metastasis, we developed a microfluidic in vitro assay incorporating 3D ECM scaffolds that emulate in vivo tumor microenvironment in 3D. We cultured highly invasive breast cancer cell, MDA-MB-231, and non-invasive breast cancer cell, MCF-7, together in a mixed population and let the cells interact with other cells and ECMs.

As a result, we found that heterogeneity in cancer cell population promotes metastatic potential. Highly invasive breast cancer cells, MDA-MB-231, made microtracks in ECM and leaded collective migration of non-invasive breast cancer cells, MCF-7, into the microtracks. When MCF-7 were cultured alone, MCF-7 cells didn't invade into ECM. It has been widely announced that only stromal cells such as fibroblast remodel ECM and enhance the invasion potential of cancer cells. However, from the result of our experiment, cancer cells that have different characteristics in single tumor can promote the metastatic potential, result from that ECM remodeling (generating microtracks) by highly invasive cancer cells. It was precisely investigated by IF, rt-PCR and TEM analysis in microfluidic environments. Understanding about not only genetic and molecular definition of cancer heterogeneity, but also the role of heterogeneous cancer cell population in metastatic dissemination, could help new drugs and therapeutic strategies development.

Citation Format: Yoojin Shin, Hyo Eun Jeong, Sewoon Han, Jihee Won, Seok Chung. The role of cancer heterogeneity in 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 C13.