bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A43: Direct visualization of changes of lymphatic function and drainage pathways in lymph node metastasis of B16F10 melanoma using near-infrared fluorescence imaging
Authors: Kwon Sunkuk , Agollah Germaine , Wu Grace , Sevick-Muraca Sevick
Published: August 2014

The lymphatic system provides an initial route for cancer cell dissemination in many cancers including melanoma. However, it is largely unknown how the lymphatic system changes during tumor progression, due in part to the lack of imaging techniques available. In this study, we non-invasively imaged changes of lymphatic function and drainage patterns using dynamic near-infrared fluorescence (NIRF) imaging.

Dynamic NIRF imaging following intradermal injection of indocyanine green (ICG) was conducted in C57BL/6 mice prior to inoculation of B16F10 murine melanoma cells to the dorsal foot for baseline data and until 21 days post-implant (p.i.). A series of acquired fluorescent images were quantified to measure lymphatic contractile function. Computed tomography (CT) was also performed to measure the volume of tumor-draining lymph nodes (LNs). In addition, intravital color images using a stereomicroscope were acquired after a skin incision. Then, the PLNs and inguinal LNs (ILNs) were harvested for ex vivo NIRF and intravital color imaging.

We observed significant reduction of lymphatic contractility from 7 days p.i. until 21 days p.i.. Altered lymphatic drainage patterns were detected at 21 days p.i., due to lymphatic obstruction of normal lymphatic drainages caused by extensive tumor invasion of draining LNs. Magnified fluorescent images of tumor-draining lymphatic vessels showed numerous fluorescent lymphatic vessels at day 21 p.i. as compared to well-defined fluorescent vessels from a baseline image. Our CT data showed enlarged tumor-draining PLNs at 21 days p.i. and intravital images confirmed the PLN harbored melanoma cells. Overlay of white light and fluorescent images of dissected LNs showed strong ICG fluorescence in the ILN and to less extent in the PLN on the tumor bearing side, indicating significantly reduced lymph flow to the PLN due to blockage of normal lymph flow by tumor in the PLN.

Since lymphatic function and architecture were progressively altered during tumor growth and metastasis, non-invasive NIRF imaging may provide a new method to stage disease. In addition, this novel technique can be used as a diagnostic method to non-invasively assess lymphatic response as mechanism of therapeutic action.

Citation Format: Sunkuk Kwon, Germaine Agollah, Grace Wu, Sevick Sevick-Muraca. Direct visualization of changes of lymphatic function and drainage pathways in lymph node metastasis of B16F10 melanoma using near-infrared fluorescence imaging. [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 A43.
Abstract A47: Microfluidic in vitro platform for imaging metastasis: Perfusable microvascular networks and cancer cells on a microfluidic chip
Authors: Jeon Noo Li , Kim Sudong , Hyunjae Lee , Minhwan Chung
Published: August 2014

In vivo imaging has provided significant insights to different mechanisms involved in cancer metastasis. An in vitro experimental platform that reproduce cancer microenvironment with blood vessels, tumors, immune cells and others would allow more detailed experiments with more experimental conditions can complement the animal studies.

We describe a reproducible, in vitro approach to grow perfusable 3D microvascular networks on a microfluidic chip. These engineered blood vessels exhibit morphological and biochemical markers of tight junctions, apical-basal polarity, basement membrane deposition, and upregulated markers in response to inflammatory cues.

A novel microfluidic device was designed and tested for real-time imaging of various steps of cancer metastasis. Perfusable microvascular networks formed within the microfluidic device reproduce the 3D cellular niche, facilitating high-resolution live imaging of cancer cell extravasation. This approach provides a platform for developing physiologically complex but experimentally straightforward human disease models and holds potential for medical and pharmaceutical applications in drug screening and basic cancer biology.

Citation Format: Noo Li Jeon, Sudong Kim, Lee Hyunjae, Chung Minhwan. Microfluidic in vitro platform for imaging metastasis: Perfusable microvascular networks and cancer cells on a microfluidic chip. [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 A47.
Abstract A5: Functional role of SOX2 in hypoxia-mediated metastasis-associated functions in prostate cancer
Authors: Dai Yao , Bae Kyung-Mi , Vieweg Johannes , Siemann Dietmar W.
Published: August 2014

Purpose: SOX2 is a typical embryonic stem cell marker that is involved in not only tumorigenesis but also metastasis in several human malignancies including prostate cancer. Hypoxia has been demonstrated as a common feature of prostate cancer that is associated with disease progression and poor prognosis. Although SOX2 has been shown to be activated by oxygen depletion in other cancer types, its functional role under hypoxic microenvironment in prostate cancer setting and its association with hypoxia inducible factors (HIFs) have not been reported. In the present study, we evaluate the role of SOX2 in hypoxia-mediated cellular behaviors associated with metastasis, including invasion and sphere formation, in prostate cancer cells.

Experimental Design: A tissue microarray containing samples from 80 prostate cancer patients was employed to examine the expression of HIF-1α and its correlation with SOX2 by immunohistochemical analysis. Expression of stem cell markers and HIFs was detected by Western blot. The role of SOX2 and HIF-1/2α in the regulation of cell invasion and sphere formation with various hypoxic exposures was investigated by short hairpin RNA (shRNA) - mediated knockdown in PC-3, DU145 and LNCaP cells.

Results: HIF-1α expression was significantly elevated in prostate cancer compared to non-malignant prostate tissues, and in tumors HIF-1α was strongly correlated with SOX2. In all three prostate cancer cells, while HIF-1α and HIF-2α were mainly induced under short term (acute) and long term (chronic) hypoxic conditions, respectively, SOX2 was the only one among five stem cell markers that was stimulated by both hypoxic exposures. Genetic depletion of SOX2 attenuated capacities of tumor cells to invade under acute hypoxia and form spheroids under chronic hypoxia. In addition, knockdown of HIF-1α but not HIF-2α decreased acute hypoxia-mediated cell invasion, whereas only HIF-2α gene silencing resulted in reduced sphere formation capacity mediated by long term oxygen deprivation.

Conclusions: SOX2 is suggested to be correlated with hypoxia in prostate cancer. SOX2 plays an important role in acute hypoxia-induced invasiveness and chronic hypoxia-induced stemness that is associated with HIF-1α and HIF-2α activation, respectively. These results indicate that SOX2 may be a key mediator for hypoxia-induced metastasis-associated functions hence may serve as a potential target for therapeutic interventions for advanced prostate cancer.

Citation Format: Yao Dai, Kyung-Mi Bae, Johannes Vieweg, Dietmar W. Siemann. Functional role of SOX2 in hypoxia-mediated metastasis-associated functions in prostate 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 A5.
Abstract A74: Tumor secreted Hsp90 initiates tumor growth and the epithelial to mesenchymal transition (EMT) in prostate cancer
Authors: Hance Michael W. , Nolan Krystal , Gopal Udhayakumar , Bohonowych Jessica E. , Jezierska-Drutel Agnieszka , Neumann Carola A. , Liu Haibo , Isla Garraway P. , Franco Omar E. , Hayward Simon W. , Isaacs Jennifer S.
Published: August 2014

Prostate cancer (PCa) is the most frequently diagnosed malignancy and second most common cause of cancer related lethality in men. Disease mortality is due primarily to metastatic spread, highlighting the urgent need to identify factors involved in this progression. Increased secretion of heat shock protein 90 (Hsp90) has been reported in diverse cancer cells, and secreted, or extracellular Hsp90 (eHsp90) is also found in patient plasma. Although eHsp90 promotes cancer cell motility and invasion and facilitates tumor metastasis, the mechanistic basis for its activity has remained unknown. The goal of this study was to explore whether extracellular Hsp90 (eHsp90) may play a role in PCa progression. To explore this, 3 sets of lineage-related differentially metastatic cell pairs were utilized to evaluate eHsp90 expression and activity. We found that eHsp90 secretion correlated with PCa aggressiveness. To evaluate the functional role of eHsp90, we modulated its expression and activity. Upregulation of eHsp90 was accomplished by exposure of prostate epithelial cells to exogenous Hsp90 protein, or alternatively, cells were transduced with a lentivirus directing the secretion of Hsp90. Blockade of eHsp90 function was accomplished by exposure of cells to anti-Hsp90 antibodies or to a cell-impermeant specific inhibitor of Hsp90. We demonstrate that eHsp90 is a critical factor required for PCa cell motility and for initiation of the epithelial to mesenchymal transition (EMT) genetic program. Moreover, modest increases in eHsp90 were sufficient to induce tumor growth and localized invasion in a subrenal capsule xenograft model of PCa. Finally, eHsp90 was detected in human cancer specimens and its expression was correlated with upregulated expression of pro-invasive genes. Our findings offer insights into the mechanistic basis of eHsp90 in PCa, and further support a putative clinical role for eHsp90 as a potential driver of PCa progression.

Citation Format: Michael W. Hance, Krystal Nolan, Udhayakumar Gopal, Jessica E. Bohonowych, Agnieszka Jezierska-Drutel, Carola A. Neumann, Haibo Liu, Garraway P. Isla, Omar E. Franco, Simon W. Hayward, Jennifer S. Isaacs. Tumor secreted Hsp90 initiates tumor growth and the epithelial to mesenchymal transition (EMT) in prostate 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 A74.
Abstract A75: Circulating tumor cells in human breast cancer exhibit dynamic changes in epithelial and mesenchymal composition
Authors: Yu Min , Bardia Aditya , Wittner Ben , Stott Shannon , Smas Malgorzata , Ting David , Isakoff Steven , Ciciliano Jordan , Wells Marissa , Shah Ajay , Concannon Kyle , Donaldson Maria , Sequist Lecia , Brachtel Elena , Sgroi Dennis , Baselga Jose , Ramaswamy Sridhar , Toner Mehmet , Haber Daniel , Maheswaran Shyamala
Published: August 2014

Epithelial mesenchymal transition (EMT) has been postulated to contribute to the dissemination of cancer cells, but supporting histopathological evidence is limited. We used a microfluidic device to isolate circulating tumor cells (CTCs), combined with multiplex RNA-in-situ hybridization (ISH) and RNA sequencing, to quantify and characterize EMT in breast CTCs. Whereas only rare cells in the primary tumor expressed both mesenchymal and epithelial markers, such biphenotypic as well as purely mesenchymal cells were enriched among CTCs, across all histological subtypes of breast cancer. Analysis of treatment responses in 10 patients suggested an association of mesenchymal CTCs with disease progression. In an index patient followed longitudinally, fluctuation in epithelial and mesenchymal states was observed as a function of initial response and subsequent resistance to therapy. Mesenchymal markers were predominant in clusters of tumor cells, many of which had adherent platelets. Finally, RNA sequencing of CTC clusters identified EMT-related signatures, including FOXC1, a known transcriptional regulator of EMT. Together, these data support a role for EMT in the blood-borne dissemination of breast cancer and point to the dynamic nature of this cell fate change.

Citation Format: Min Yu, Aditya Bardia, Ben Wittner, Shannon Stott, Malgorzata Smas, David Ting, Steven Isakoff, Jordan Ciciliano, Marissa Wells, Ajay Shah, Kyle Concannon, Maria Donaldson, Lecia Sequist, Elena Brachtel, Dennis Sgroi, Jose Baselga, Sridhar Ramaswamy, Mehmet Toner, Daniel Haber, Shyamala Maheswaran. Circulating tumor cells in human breast cancer exhibit dynamic changes in epithelial and mesenchymal composition. [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 A75.
Abstract A85: Novel mouse models to investigate the molecular pathogenesis of metastatic osteosarcoma
Authors: Zhao Shuying , Kurenbekova Lyazat , Donehower Lawrence A. , Yustein Jason T.
Published: August 2014

Background: Osteosarcoma (OS) is the primary bone tumor in the pediatric population with the main determinant for patient prognosis being the presence of metastatic disease. Approximately 25-30% of patients will present with metastatic osteosarcoma. Patients with metastatic disease have long-term survival rates often <20%.

Objectives: We hypothesize that the molecular pathogenesis of metastatic osteosarcoma is different from localized disease. We intend this work to provide a relevant, endogenous model of metastatic OS that can be utilized to advance our understanding of the molecular pathogenesis of the disease, insights into novel therapeutic targets and as a pre-clinical model for investigating the efficacy of novel therapies.

Design: We have developed a tissue-specific alteration of the p53 status by using osteoblast specific Cre-recombinase expressing mice to generate progeny that spontaneously form endogenous osteosarcomas. Through the use of a mutated, gain of function form of p53, shown previously to be associated with metastatic disease, we have developed a novel immunocompetent model that significantly enhances the endogenous development of metastatic OS.

Results: Tumor analysis has revealed genetic insights in the metastatic progression of osteosarcoma. These include the significant downregulation of Wnt-signaling inhibitors, such as NKD2, APCDD1 and Wnt5a in the metastatic tumors. Functional studies have determined that overexpression of NKD2, also downregulated in several human OS metastatic cell lines, in metastatic mouse OS cell lines leads to a significant decrease in metastatic lung lesions upon transplantation into immunodeficient mice. Possible NKD2-dependent functions include regulation of not only the Wnt signaling pathway, but also blood vessel formation, regulation of cell migration and cell adhesion.

We have also noted the dysregulation of several critical microRNAs in metastatic OS, including the upregulation of mir-130b in the metastatic lesions. This particular microRNA has recently been shown to have clinical correlation in Ewing's sarcoma, with higher levels of tumor mir-130b having a significantly poorer outcome. We are actively pursuing the role of this (and other) microRNA(s) in the molecular pathogenesis of metastatic osteosarcoma.

Conclusion: This novel model has enabled valuable molecular insights into the development and progression of metastatic OS that can lead to the identification of novel therapeutic targets.

Citation Format: Shuying Zhao, Lyazat Kurenbekova, Lawrence A. Donehower, Jason T. Yustein. Novel mouse models to investigate the molecular pathogenesis of metastatic osteosarcoma. [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 A85.
Abstract A97: Antitumor effect of metformin in breast cancer is associated with AMPK and FOXO3a activation
Authors: Fonseca Eveline A I , Oliveira Maria Aparecida , Eichler Rosangela , Akamine Eliana H. , Carvalho Maria Helena C. , Barbosa Aneli M. , Dekker Robert F. H. , Khaper Neelam , Fortes Zuleica B.
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 (PR15) of the Conference Proceedings.

Citation Format: Eveline A I Fonseca, Maria Aparecida Oliveira, Rosangela Eichler, Eliana H. Akamine, Maria Helena C. Carvalho, Aneli M. Barbosa, Robert F. H. Dekker, Neelam Khaper, Zuleica B. Fortes. Antitumor effect of metformin in breast cancer is associated with AMPK and FOXO3a activation. [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 A97.
Abstract B102: Fibroblast-dependent modulation of drug sensitivity, stemcellness, and tumor cell dormancy
Authors: Paulsson Janna , Strell Carina , Roswall Pernilla , Rannikmae Helena , Weiland Anne , Rydén Lisa , Östman Arne
Published: August 2014

Solid tumors are composed of tumor cells and different cell types making up the tumor stroma — endothelial cell, pericytes, immune cells and cancer associated fibroblasts. We focus on the impact of cancer associated fibroblasts on the stem cell properties of tumor cells, including drug sensitivity, anchorage independent growth and tumor initiating capacities. Previous work in our group showed that expression of platelet derived growth factor receptor β (PFGFRβ) in the tumor stroma is associated with worse prognosis in breast cancer. Furthermore, two experimental approaches were used to explore PDGFR-dependent paracrine signaling: (1) primary human breast fibroblasts isolated from normal as well as cancer tissue co-cultured with human breast cancer cell lines and (2) tumor cells, cancer associated fibroblasts and normal breast tissue fibroblasts derived from a genetic breast cancer mouse model.

When looking at PFGFRβ expression in tumors from a randomized tamoxifen study we found that tumors with high expression in the tumor stroma did not seem to respond to the treatment. Ongoing in vitro studies are analyzing the impact of co-cultured fibroblasts on breast cancer cell tamoxifen sensitivity in vitro. Specifically we want to investigate how PDGF receptors, expressed by the tumor stroma, play a role in the sensitivity to tamoxifen by the tumor cells.

We have also investigated the impact of fibroblasts on stemness properties of tumor cells derived from tumors of the MMTV polyoma middle T antigen driven mouse breast cancer model. Conditioned medium of tumor associated fibroblasts but also from normal mouse breast tissue fibroblasts increased the anchorage independent sphere forming capacity of the tumor cells in vitro. Conditioned media from the tumor cells themselves had similar effects. Tumor-supporting effects of fibroblasts were confirmed after orthotopic coinjection of tumor cells together with fibroblasts in syngeneic mice. The presence of fibroblasts resulted in a significant increase in the tumor take rate.

Together these studies identify fibroblast-mediated modulation of “stemness” of breast cancer cells and a PDGFR-dependent modulation by fibroblasts of tamoxifen-sensitivity.

Citation Format: Janna Paulsson, Carina Strell, Pernilla Roswall, Helena Rannikmae, Anne Weiland, Lisa Rydén, Arne Östman. Fibroblast-dependent modulation of drug sensitivity, stemcellness, and tumor cell dormancy. [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 B102.
Abstract B2: Oligodendritic precursor mediates vascular remodeling within glioma microenvironment in PDGF-C dependent manner
Authors: Huang Yujie , Lyden David , Greenfield Jeffrey
Published: August 2014

The platelet derived growth factor (PDGF) signaling pathway is upregulated in many glioma subtypes, plays a role in gliomagenesis and promotes brain tumor progression. In this study, we demonstrate a role for PDGF signaling in the maintenance of brain tumor microenvironment homeostasis. We detected PDGFRa-expressing stromal cells potentially derived from oligodendritic precursor cells in both human and mouse high-grade gliomas. PDGFRa+ cells are predominately located at the invasive front of gliomas, and have a unique perivascular distribution. We suggest that these stromal cells play an important role in both initiating angiogenesis and supporting tumor progression. In an orthotopic model of glioma in PDGFRa deficient mice, tumor growth was significantly inhibited and gadolinium enhancement was decreased shown by bioluminescence and MRI, respectively. Glioma-associated blood vessels in PDGFRa knockout mice have smaller lumens and relatively normal vascular morphology. Glioma-associated oligodendritic precursor cells promoted endothelial sprouting and tubule formation by abrogating the inhibitory effect of astrocytes. We also demonstrated stromal-derived PDGF-CC as a key factor in the recruitment and activation of PDGFRa+ stromal cells. Utilizing PDGF-CC deficient mice, we recapitulate the glioma phenotype demonstrated in PDGFRa; knockout mice, suggesting their joint contribution to this pathway's integrity. In conclusion, the PDGF-CC/PDGFRa signaling axis in glioma stroma appears to contribute to the remodeling of the vascular architecture in the brain and influences subsequent angiogenesis.

Citation Format: Yujie Huang, David Lyden, Jeffrey Greenfield. Oligodendritic precursor mediates vascular remodeling within glioma microenvironment in PDGF-C dependent manner. [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 B2.
Abstract B24: Hepatocyte growth factor activator inhibitor type 1 (HAI-1) suppresses tumor growth and metastasis of pancreatic adenocarcinoma cells
Authors: Ye Jing-Jia , Kawaguchi Makiko , Haruyama Yukihiro , Kanemaru Ai , Fukushima Tsuyoshi , Kataoka Hiroaki
Published: August 2014

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a membrane-bound serine proteinase inhibitor expressed on epithelial and carcinoma cell surface. HAI-1 regulates hepatocyte growth factor activator, kallikrein 1-related peptidase 4 and 5 and several membrane-anchored serine proteinases such as matriptase, prostasin, hepsin, TMPRSS13 and human airway trypsin-like protease. These proteases are reported to be involved in tumor progression, and among them, matriptase and prostasin are known to activate protease-activated receptor-2 (PAR-2). Previously, we reported that S2-CP8 cells, a metastatic variant of SUIT-2 pancreatic adenocarcinoma cells, showed decreased HAI-1 expression compared to SUIT-2 and acquired EMT phenotype. To assess the HAI-1 function in metastasis of S2-CP8 cells, we established a stable S2-CP8 subline that expressed HAI-1 under control of tetracycline-regulated promoter and analyzed in a nude mouse orthotopic xenograft model.

S2-CP8 cells expressed matriptase, TMPRSS13, TMPRSS4, prostasin and PAR-2. Matrigel invasion assay revealed that S2-CP8 cells showed enhanced invasion compared to SUIT-2 cells. The enhanced invasiveness was significantly suppressed by forced HAI-1 expression. Moreover, the HAI-1 loss-induced invasiveness was partly alleviated by knockdown of matriptase or PAR-2. Then, we examined the effect of HAI-1 expression on metastasis in vivo using an orthotopic xenograft model. Four weeks after the transplantation, mice were sacrificed and prepared for histological evaluation. We found that 50% of control mice developed distant metastasis. However, mice treated with tetracycline to express HAI-1 did not develop the metastasis. Though there was no difference in cell proliferation rates in vitro, tumor size was decreased by the expression of HAI-1. These results suggested that, in SUIT-2 pancreatic adenocarcinoma cell line, loss of HAI-1 enhances cell invasiveness, at least in part, through PAR-2/matriptase system in vitro, and HAI-1 plays a suppressive role in metastasis of the cells in vivo.

Citation Format: Jing-Jia Ye, Makiko Kawaguchi, Yukihiro Haruyama, Ai Kanemaru, Tsuyoshi Fukushima, Hiroaki Kataoka. Hepatocyte growth factor activator inhibitor type 1 (HAI-1) suppresses tumor growth and metastasis of pancreatic adenocarcinoma 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 B24.
Abstract B3: CD24 affects tumor cell properties and cytokine secretion in a STAT3-dependent fashion
Authors: Bretz Niko P. , Salnikov Alexei V. , Moldenhauer Gerhard , Altevogt Peter
Published: August 2014

CD24 is a glycosyl-phosphatidylinositol (GPI)-anchored membrane protein frequently over-expressed in human carcinomas and associated with metastasis and poor prognosis. Previous work has shown that CD24 expression affects various cellular properties such as invasion, adhesion, proliferation and apoptosis in vitro as well as tumor growth in vivo.

By analyzing underlying mechanisms of CD24-dependent tumor cell behaviour we discovered the influence of CD24 on STAT3 signaling and gene regulation via the tyrosine kinase c-Src.

Altered activity of c-Src mediated by CD24 knock-down was responsible for reduced levels of phosphorylated STAT3 (Y705). Diminished activity of STAT3 was confirmed by luciferase-reporter assays. Silencing of c-Src, similar to CD24, reduced the expression of prototype STAT3-regulated genes. Likewise, the over-expression of CD24 augmented c-Src activity, the recruitment of c-Src into lipid rafts, levels of p-STAT3 and the expression of STAT3-dependent target genes. Functionally, depletion of CD24 reduced cell proliferation and increased apoptosis, suggesting a pivotal role of regulated target genes for these functional effects.

Even more profoundly, CD24 expression was accompanied with the secretion of the STAT3 target IL-6. We could show a strong correlation between IL-6 secretion and CD24 expression in tumor cell lines. Furthermore, treatment with conditioned cell culture medium from CD24 over-expressing- or CD24 knock-down tumor cells initiated paracrine STAT3 signaling and gene regulation in the monocytic precursor cell line THP-1. This suggested a regulative role of CD24 on the tumor microenvironment. We propose that CD24 is capable of influencing not only tumor cellular properties but also has impact on the cytokine milieu of the tumor microenvironment.

Citation Format: Niko P. Bretz, Alexei V. Salnikov, Gerhard Moldenhauer, Peter Altevogt. CD24 affects tumor cell properties and cytokine secretion in a STAT3-dependent fashion. [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 B3.
Abstract B44: Loss of the keratin cytoskeleton is not sufficient to induce epithelial mesenchymal transition in a novel KRAS-driven sporadic lung cancer mouse model
Authors: Koenig Katharina , Meder Lydia , Kroeger Cornelia , Diehl Linda , Florin Alexandra , Rommerscheidt-Fuss Ursula , Kahl Philip , Wardelmann Eva , Magin Thomas M. , Buettner Reinhard , Heukamp Lukas C.
Published: August 2014

Epithelial-to-mesenchymal transition (EMT), the phenotypical change of cells from an epithelial to a mesenchymal type, is thought to be a key event in invasion and metastasis of adenocarcinomas. These changes involve loss of keratin expression as well as loss of cell polarity and adhesion. We here aimed to determine whether the loss of keratin expression itself drives increased invasion and metastasis in adenocarcinomas and whether keratin loss leads to the phenotypic changes associated with EMT. Therefore, we employed a recently described murine model in which conditional deletion by Cre-recombinase leads to loss of the entire keratin

multiprotein family. These mice were crossed into a newly generated Cre-recombinase inducible KRAS-driven murine lung cancer model to examine the effect of keratin loss on morphology, invasion and metastasis as well as expression of EMT related genes in the resulting tumors.

We here clearly show that loss of a functional keratin cytoskeleton did not significantly alter tumor morphology or biology in terms of invasion, metastasis, proliferation or tumor burden and did not lead to induction of EMT. Further, tumor cells did not induce synchronously expression of vimentin, which is often seen in EMT, to compensate for keratin loss. In summary, our data suggest that changes in cell shape and migration that underlie EMT, are dependent on changes in signaling pathways that cause secondary changes in keratin expression and organization. Thus, we conclude that that loss of the keratin cytoskeleton per se is not sufficient to causally drive EMT in this tumor model.

Citation Format: Katharina Koenig, Lydia Meder, Cornelia Kroeger, Linda Diehl, Alexandra Florin, Ursula Rommerscheidt-Fuss, Philip Kahl, Eva Wardelmann, Thomas M. Magin, Reinhard Buettner, Lukas C. Heukamp. Loss of the keratin cytoskeleton is not sufficient to induce epithelial mesenchymal transition in a novel KRAS-driven sporadic lung cancer mouse 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 B44.
Abstract B46: The role of integrin switching and Deptor in metastatic breast cancer
Authors: Parvani Jenny G. , Taylor Molly A. , Schiemann William P.
Published: August 2014

Mammary tumorigenesis converts TGF-β; from a tumor suppressor in normal mammary epithelial cells (MECs) to a tumor promoter in cancerous MECs, likely via Epithelial-Mesenchymal Transiton (EMT). Integrin signaling plays a critical role in this conversion, although the underlying molecular mechanisms remain elusive. Previously, both β1 and β3 integrins have been associated with TGF-β; mediated EMT and metastasis in breast cancer, making both integrins attractive therapeutic targets. Importantly, the interplay and requirement for β1 and β3 integrins in mediating metastatic disease and the downstream effectors that effectuate the malignancy associated with these intergrins have not been fully evaluated. Here, we describe an integrin switching mechanism where functional disruption of β1 integrin mediates compensatory upregulation of β3 integrin expression, which augments TGF-β; signaling and mediates 3D outgrowth of metastatic breast cancer cells. We demonstrate that sole targeting of β1 integrin is insufficient in reducing tumor burden, while sole targeting of β3 integrin significantly alleviates the disease. Furthermore, we identify Deptor, the endogenous inhibitor of mTOR, to be downregulated in β3 integrin expressing cells, suggesting that attenuated Deptor expression may engender the malignancy associated with β3 integrin. Importantly, functional disruption of Deptor in systemically invasive 4T07 cells induces their acquisition of mesenchymal characteristics, while attenuating their ability to proliferate. Collectively, we demonstrate that targeting β1 integrin alone is insufficient in alleviating metastatic breast cancer, while sole targeting of β3 integrin significantly attenuates the disease. Furthermore, our data identifies Deptor as a potential downstream effector in β3 integrin-dependent malignancies.

Citation Format: Jenny G. Parvani, Molly A. Taylor, William P. Schiemann. The role of integrin switching and Deptor in metastatic 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 B46.
Abstract B49: Integrin alpha2beta1 (α2β1) promotes prostate cancer skeletal metastasis
Authors: Sottnik Joseph L. , Daignault-Newton Stephanie , Zhang Xiaotun , Colm Morrissey , Hussain Maha H. , Keller Evan T. , Hall Christopher L.
Published: August 2014

Men who die of prostate cancer (PCa) do so because of systemic metastases, the most frequent of which are within the skeleton. Recent data suggest that the colonization of the skeleton is mediated in part by collagen type I, the most abundant protein within the bone. We have shown that enhanced collagen I binding through increased expression of integrin α2β1 stimulated in vitro invasion and promoted the growth of PCa cells within the bone. Accordingly, we tested whether α2β1 integrin is both a mediator and a predictive marker of skeletal metastasis. To directly test whether α2β1 integrin expression mediates PCa cell metastasis, α2 integrin was over-expressed in low-tumorigenic LNCaP PCa cells or selectively knocked-down in highly metastatic LNCaPcol PCa cells. The over-expression of full-length cDNA to α2 integrin stimulated whereas shRNA-mediated knock-down of α2 integrin abolished the ability of transduced cells to bind to or migrate towards collagen I. Correspondingly, α2 integrin knock-down reduced both the tumor volume of intra-osseous lesions and the formation of experimental metastases in the liver and skeleton compared to control-transduced cells demonstrating that α2β1 promotes the establishment of distant metastases. To investigate the clinical significance of α2β1 expression in PCa, α2β1 protein was measured in prostatic tissues and in soft tissue and bone metastases. The data demonstrate that α2β1 protein was elevated in PCa skeletal metastases compared to either PCa primary lesions or soft tissue metastases suggesting that α2β1 mediates the selective metastasis of PCa cells to the bone. Taken together, these data support that α2β1 integrin promotes the systemic metastasis of PCa cells and provide evidence that α2β1 integrin activity may promote skeletal metastasis in PCa patients. These studies provide a strong rationale for the development of α2β1 as a therapeutic target for the treatment of PCa skeletal metastases and thus have the potential to reduce both the incidence and mortality associated with metastatic PCa.

Citation Format: Joseph L. Sottnik, Stephanie Daignault-Newton, Xiaotun Zhang, Morrissey Colm, Maha H. Hussain, Evan T. Keller, Christopher L. Hall. Integrin alpha2beta1 (α2β1) promotes prostate cancer skeletal 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 B49.
Abstract B55: The regulation of matriptase activation through PDGF D-mediated extracellular acidosis
Authors: Najy Abdo J. , Kim Hyeong-Reh C.
Published: August 2014

Prostate cancer (PCa) is the second most diagnosed and sixth leading cause of cancer-related death in men worldwide. Activation of β-PDGFR (platelet-derived growth factor receptor) has been demonstrated in 50% of PCa cases and it is part of a 5-gene signature predicting PCa recurrence after prostatectomy. Analysis of the β-PDGFR ligands in PCa revealed association between PDGF D expression and Gleason score as well as tumor stage. When we examined the functional consequences of PDGF ligand-specific β-PDGFR signaling in PCa, we discovered a novel PDGF D-specific autocrine pathway, resulting in activation/shedding of the serine protease matriptase leading to PCa cell invasion, migration, and tumorigenesis. The present study showed that PDGF D, not PDGF B, induces extracellular acidification which correlates with increased matriptase activation. Furthermore, exposure of control or PDGF B expressing cells to an acidic buffer resulted in increased matriptase activation, indicating the functional significance of the acidic milieu in matriptase activation. A cDNA microarray analysis showed that PDGF D/β-PDGFR signaling upregulates expression of the acidosis regulator carbonic anhydrase IX (CA IX), a classic target of the transcriptional factor HIF-1. Interestingly, cellular fractionation displayed a strong HIF-1 nuclear localization in PDGF D expressing cells. Treatment of vector control or PDGF B expressing cells with the HIF-1 activator, CoCl2, specifically led to increased CA IX expression accompanied by extracellular acidosis and matriptase activation. Taken together, these novel findings reveal a new paradigm in matritpase activation involving PDGF D-specific signal transduction leading to extracellular acidosis. Considering the fact that intratumoral acidosis leads to chemo- and radiation resistance, understanding the role PDGF signaling plays in this process will enhance the efficacy of current clinical therapeutics.

Citation Format: Abdo J. Najy, Hyeong-Reh C. Kim. The regulation of matriptase activation through PDGF D-mediated extracellular acidosis. [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 B55.
Abstract C24: Identification and characterization of serum biomarkers associated with metastatic progression of breast cancer
Authors: Za'abi Adhari Abdullah Al , Graves Steven , Hansen Marc
Published: August 2014

Breast cancer is the most prevalent form of cancer among women in the United States. Treatment is greatly complicated by metastasis of cells from the primary tumor site, which increases mortality; about 40 percent of breast cancer patients will relapse and develop metastatic disease. Eighty percent of breast cancer patients are treated with adjuvant chemotherapy to decrease their risk of getting metastasis, while as indicated previously only 40% of them will eventually relapse and develop metastatic disease. Despite this precautionary measure, adjuvant chemotherapy is shown to increase 15-years survival of such patients by only 3-10%. Further, about 40,000 women die of breast cancer every year due to undetected early metastasis. Clearly detection of metastatic disease has major clinical implications. Identifying specific blood serum biomarkers that accurately and reproducibly indicate the presence of metastatic disease could greatly improve treatment, as they could substitute for more invasive procedures that are in current use and be used to monitor remission at higher resolution. Analysis of protein content in serum has been widely used as a method for biomarker identification, but detection, identification, and analysis of short peptide biomarkers faces technical challenges that have prevented widespread application of this approach. We report here results from a proteomics study aimed at identifying and characterizing short peptide biomarkers in serum samples taken from patients with stage I and stage III breast cancer. Serum samples contain over 2500 distinct peaks representing distinct peptides. A preliminary analysis reveals that the abundance of 92 peptides are significantly reduced in samples from patients with stage III disease, while the abundance of 64 peptides show significant increase in stage III sera. A large number of additional peptides show smaller changes in abundance in sera from the two patient groups. Our future work will be to characterize the identity of the serum biomarkers identified and validate their appearance or disappearance in a large cohort of patients.

Citation Format: Adhari Abdullah Al Za'abi, Steven Graves, Marc Hansen. Identification and characterization of serum biomarkers associated with metastatic progression of 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 C24.
Abstract C34: WAVE3 is required for TGF-β-mediated EMT
Authors: Taylor Molly A. , Davuluri Gangarao , McCue Brian , Plow Edward F. , Schiemann William P. , Sossey-Alaoui Khalid
Published: August 2014

Breast cancer (BC) is the second leading cause of cancer death in women in the United States. Metastasis accounts for the death of ~90% of these patients, yet the mechanisms underlying this event remain poorly defined. WAVE3 belongs to the WASP/WAVE family of actin-binding proteins that mediate essential roles in regulating cell shape/morphology, actin polymerization and cytoskeleton remodeling, and cell motility and invasion. Accordingly, we have shown that WAVE3 promotes the acquisition of invasive and metastatic phenotypes by human BCs. Along these lines, we also established that members of the miR-200 family, together with miR-31 suppress WAVE3 expression, thereby limiting its prometastatic behavior. We show here that Transforming Growth Factor-beta (TGFβ;) robustly decreases miR-31 expression and induces WAVE3 expression through a Smad2 and β3-integrin dependent mechanism. Moreover, WAVE3 expression is required for BC cells to undergo epithelial-mesenchymal transition (EMT) in response to TGF-β;. Accordingly, stable depletion of WAVE3 expression in metastatic MDA-MB-231 or 4T1 BC cells prevented TGF-β; from inducing the EMT as determined by monitoring changes in the actin cytoskeleton and expression of EMT markers by real-time PCR. Along these lines, WAVE3 depletion significantly inhibited the ability of 4T1 cells to migrate and proliferate in response to TGF-β;. Moreover, WAVE3 deficiency abrogated the outgrowth of MDA-MB-231 and 4T1 cell organoids in 3D organotypic cultures. In vivo bioluminescent imaging indicated that WAVE3 knockdown also decreased tumor growth and metastasis in 4T1 tumors that possess enhanced autocrine TGF-β; signaling. Collectively, these findings indicate that WAVE3 activity is required for TGF-β;-mediated EMT and suggest that measures capable of inactivating WAVE3 may alleviate BC metastasis stimulated by TGF-β;.

Citation Format: Molly A. Taylor, Gangarao Davuluri, Brian McCue, Edward F. Plow, William P. Schiemann, Khalid Sossey-Alaoui. WAVE3 is required for TGF-β-mediated EMT. [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 C34.
Abstract C42: MLKs regulate the activities of the small GTPases, Rho and Rac, to drive both single cell and collective cell migration in breast cancer invasion
Authors: Rattanasinchai Chotirat , Misek Sean , Chen Jian , Gallo Kathleen A.
Published: August 2014

Most mortality of breast cancer patients is caused by metastasis to vital organs. One of the key steps of breast cancer metastasis is invasion through the basement membrane, which is controlled by several signaling cascades including small GTPases, Rho and Rac. Mixed lineage kinases (MLKs) are a family of MAP3Ks that activate multiple MAPK pathways, including the c-Jun N-terminal kinase (JNK) pathway. Our lab has determined that MLK3-JNK signaling is critical for migration, invasion and metastasis of triple negative breast cancer cells. Herein, the effect of a small molecule pan-MLK inhibitor on tumor cell migration in 2D wound healing and in 3D dissemination in collagen type I, the most abundant collagen in the body, was determined. Cancer cells can utilize distinct modes of migration. MDA-MB-231 and 4T1 cells were chosen as models for the study of single and collective cell migration, respectively. In addition, the effect of MLK3 on Rho-GTP and Rac-GTP levels was determined. Data from this study demonstrate that an MLK inhibitor is sufficient to block migration of multiple breast cancer cell lines in wound healing assays. Furthermore, this inhibitor dramatically reduces the dissemination of cancer cell lines that utilize single, as well as, collective cell migration. Silencing of MLK3 results in enhanced Rho activity and decreased Rac activity, as well as defective focal adhesion turnover. Inhibition of the activity of MLKs or the downstream MAPK, JNK, also increases Rho activity, which is accompanied by an increase in the number and/or size of focal adhesions and an increase in actin stress fibers. Consistent with the idea that MLK inhibition reduces migration through relieving Rho suppression, addition of a Rho/ROCK inhibitor, Y-27632, partially rescues the migratory defect of MLK inhibition by reducing peripheral focal adhesions and actin stress fibers. Taken together, these data suggest that MLK3 could play a role in controlling the balance between Rac and Rho activity required for cancer cell migration and invasion. Thus, MLK inhibitors could be therapeutically useful in the context of breast cancer metastasis.

Citation Format: Chotirat Rattanasinchai, Sean Misek, Jian Chen, Kathleen A. Gallo. MLKs regulate the activities of the small GTPases, Rho and Rac, to drive both single cell and collective cell migration in breast cancer invasion. [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 C42.
Abstract C47: Assessing the role of Wnt secretion in prostate cancer skeletal metastasis
Authors: Valkenburg Kenneth C. , Williams Bart O.
Published: August 2014

Aberrant Wnt signaling is associated with prostate cancer tumorigenesis, progression, and bone metastasis. Bone metastasis is responsible for substantial morbidity and mortality in patients with advanced prostate cancer. No curative treatment currently exists for patients with bone metastasis. Wnt signaling plays an integral role in the bone-tumor environment during prostate cancer bone metastasis, particularly osteoblastic lesion formation, but the cell type that secretes Wnt ligands in this process is unknown. This knowledge would result in improved targeting strategies for chemotherapeutics. We hypothesize that Wnt ligand secretion by prostate tumor cells is necessary to establish and/or mediate progression of prostate tumor growth and associated bone formation within the skeleton. We targeted two proteins that are essential for Wnt secretion. Porcupine (Porcn) is an acyltransferase responsible for adding fatty acids to Wnt ligands, which precedes their interaction with the chaperone protein Wntless (Wls). Both Porcn and Wls are essential for Wnt ligand secretion. Without secretion, Wnt ligands are retained inside Wnt-producing cells, and therefore cannot activate Wnt receptors in an autocrine or paracrine manner. Using shRNA, we created stable Wls knockdown C42B4 prostate cancer cell lines and confirmed knockdown via quantitative PCR. We saw that when Wls is knocked down by approximately 95%, the Wnt target gene Axin2 decreased in expression by approximately 50%. This indicates that autocrine signaling is occurring in these cells, supporting our hypothesis. We have also generated three xenograft models in immune-deficient Nod-Scid-ILR2gamma-deficient (NSG) mice and are currently assessing the ability of Wls-deficient cells to metastasize and establish tumors in bone. In addition, we will treat mice injected with prostate cancer cells with a chemical Porcn inhibitor to determine the therapeutic value of such inhibitors in treating advanced prostate cancer. Given that Porcn inhibitors are in early stage human clinical trials for other cancers, our work could provide support for using said inhibitors for treatment of patients with advanced prostate cancer.

Citation Format: Kenneth C. Valkenburg, Bart O. Williams. Assessing the role of Wnt secretion in prostate cancer skeletal 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 C47.