Tumor Cell Intravasation Research Articles

LIM-homeobox gene 2 promotes tumor growth and metastasis by inducing autocrine and paracrine PDGF-B signaling

An epithelial-mesenchymal transition (EMT) is a critical process during embryonic development and the progression of epithelial tumors to metastatic cancers. Gene expression profiling has uncovered the transcription factor LIM homeobox gene 2 (Lhx2) with up-regulated expression during TGFβ-induced EMT in normal and cancerous breast epithelial cells. Loss and gain of function experiments in transgenic mouse models of breast cancer and of insulinoma in vivo and in breast cancer cells in vitro indicate that Lhx2 plays a critical role in primary tumor growth and metastasis. Notably, the transgenic expression of Lhx2 during breast carcinogenesis promotes vessel maturation, primary tumor growth, tumor cell intravasation and metastasis by directly inducing the expression of platelet-derived growth factor (PDGF)-B in tumor cells and by indirectly increasing the expression of PDGF receptor-β (PDGFRβ) on tumor cells and pericytes. Pharmacological inhibition of PDGF-B/PDGFRβ signaling reduces vessel functionality and tumor growth and Lhx2-induced cell migration and cell invasion. The data indicate a dual role of Lhx2 during EMT and tumor progression: by inducing the expression of PDGF-B, Lhx2 provokes an autocrine PDGF-B/PDGFRβ loop required for cell migration, invasion and metastatic dissemination and paracrine PDGF-B/PDGFRβ signaling to support blood vessel functionality and, thus, primary tumor growth.

The Function of Vacuolar ATPase (V-ATPase) a Subunit Isoforms in Invasiveness of MCF10a and MCF10CA1a Human Breast Cancer Cells

The vacuolar H(+) ATPases (V-ATPases) are ATP-driven proton pumps that transport protons across both intracellular and plasma membranes. Previous studies have implicated V-ATPases in the invasiveness of various cancer cell lines. In this study, we evaluated the role of V-ATPases in the invasiveness of two closely matched human breast cancer lines. MCF10a cells are a non-invasive, immortalized breast epithelial cell line, and MCF10CA1a cells are a highly invasive, H-Ras-transformed derivative of MCF10a cells selected for their metastatic potential. Using an in vitro Matrigel assay, MCF10CA1a cells showed a much higher invasion than the parental MCF10a cells. Moreover, this increased invasion was completely sensitive to the specific V-ATPase inhibitor concanamycin. MCF10CA1a cells expressed much higher levels of both a1 and a3 subunit isoforms relative to the parental line. Isoforms of subunit a are responsible for subcellular localization of V-ATPases, with a3 and a4 targeting V-ATPases to the plasma membrane of specialized cells. Knockdown of either a3 alone or a3 and a4 together using isoform-specific siRNAs inhibited invasion by MCF10CA1a cells. Importantly, overexpression of a3 but not the other a subunit isoforms greatly increased the invasiveness of the parental MCF10a cells. Similarly, overexpression of a3 significantly increased expression of V-ATPases at the plasma membrane. These studies suggest that breast tumor cells employ particular a subunit isoforms to target V-ATPases to the plasma membrane, where they function in tumor cell invasion.

Effects on tumor development and metastatic dissemination by the NKG2D lymphocyte receptor expressed on cancer cells

The stimulatory NKG2D lymphocyte receptor together with its tumor-associated ligands enable the immune system to recognize and destroy cancer cells. However, with dynamic changes unfolding, cancers exploit NKG2D and its ligands for immune evasion and suppression. Recent findings have added yet another functional dimension wherein cancer cells themselves coopt NKG2D for their own benefit to complement the presence of its ligands for self stimulation of parameters of tumorigenesis. Those findings are here extended to in vivo tumorigenicity testing by employing orthotopic xenotransplant breast cancer models in mice. Using human cancer lines with ectopic NKG2D expression and RNAi-mediated protein depletion among other controls, we show that NKG2D self-stimulation has tumor promoting capacity. NKG2D signals had no notable effects on cancer cell proliferation and survival but acted at the level of angiogenesis, thus promoting tumor growth, tumor cell intravasation and dissemination. NKG2D-mediated effects on tumor initiation may represent another factor in the observed overall enhancement of tumor development. Altogether, these results may impact immunotherapy approaches, which currently do not account for such NKG2D effects in cancer patients and thus could be misdirected as underlying assumptions are incomplete.

Macrophage contact induces RhoA GTPase signaling to trigger tumor cell intravasation

Most cancer patients die as a result of metastasis, thus it is important to understand the molecular mechanisms of dissemination, including intra- and extravasation. Although the mechanisms of extravasation have been vastly studied in vitro and in vivo, the process of intravasation is still unclear. Furthermore, how cells in the tumor microenvironment facilitate tumor cell intravasation is still unknown. Using high-resolution imaging, we found that macrophages enhance tumor cell intravasation upon physical contact. Macrophage and tumor cell contact induce RhoA activity in tumor cells, triggering the formation of actin-rich degradative protrusions called invadopodia, enabling tumor cells to degrade and break through matrix barriers during tumor cell transendothelial migration. Interestingly, we show that macrophage-induced invadopodium formation and tumor cell intravasation also occur in patient-derived tumor cells and in vivo models, revealing a conserved mechanism of tumor cell intravasation. Our results illustrate a novel heterotypic cell contact mediated signaling role for RhoA, as well as yield mechanistic insight into the ability of cells within the tumor microenvironment to facilitate steps of the metastatic cascade.

Stromally Derived Lysyl Oxidase Promotes Metastasis of Transforming Growth Factor-β–Deficient Mouse Mammary Carcinomas

The tumor stromal environment can dictate many aspects of tumor progression. A complete understanding of factors driving stromal activation and their role in tumor metastasis is critical to furthering research with the goal of therapeutic intervention. Polyoma middle T-induced mammary carcinomas lacking the type II TGF-β receptor (PyMT(mgko)) are highly metastatic compared with control PyMT-induced carcinomas (PyMT(fl/fl)). We hypothesized that the PyMT(mgko)-activated stroma interacts with carcinoma cells to promote invasion and metastasis. We show that the extracellular matrix associated with PyMT(mgko) tumors is stiffer and has more fibrillar collagen and increased expression of the collagen crosslinking enzyme lysyl oxidase (LOX) compared with PyMT(fl/fl) mammary carcinomas. Inhibition of LOX activity in PyMT(mgko) mice had no effect on tumor latency and size, but significantly decreased tumor metastasis through inhibition of tumor cell intravasation. This phenotype was associated with a decrease in keratin 14-positive myoepithelial cells in PyMT(mgko) tumors following LOX inhibition as well as a decrease in focal adhesion formation. Interestingly, the primary source of LOX was found to be activated fibroblasts. LOX expression in these fibroblasts can be driven by myeloid cell-derived TGF-β, which is significantly linked to human breast cancer. Overall, stromal expansion in PyMT(mgko) tumors is likely caused through the modulation of immune cell infiltrates to promote fibroblast activation. This feeds back to the epithelium to promote metastasis by modulating phenotypic characteristics of basal cells. Our data indicate that epithelial induction of microenvironmental changes can play a significant role in tumorigenesis and attenuating these changes can inhibit metastasis. Cancer Res; 73(17); 5336-46. ©2013 AACR.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Tumor MMP-1 Activates Endothelial PAR1 to Facilitate Vascular Intravasation and Metastatic Dissemination

Intravasation, the active entry of primary tumor cells into the vasculature, remains the least studied step in the metastatic cascade. Protease-mediated escape and stromal invasion of tumor cells represent widely accepted processes leading up to the intravasation step. However, molecular factors that contribute directly to tumor cell vascular penetration have not been identified. In this study, the in vivo role of the collagenolytic protease, MMP-1, in cancer cell intravasation and metastasis was analyzed by using a highly disseminating variant of human HEp3 epidermoid carcinoma, HEp3-hi/diss. Although naturally acquired or experimentally induced MMP-1 deficiency substantially suppressed HEp3-hi/diss intravasation, supplementation of recombinant MMP-1 to MMP-1-silenced primary tumors restored their impaired vascular dissemination. Surprisingly, abrogation of MMP-1 production and activity did not significantly affect HEp3-hi/diss migration or matrix invasion, suggesting noncollagenolytic mechanisms underlying MMP-1-dependent cell intravasation. In support of such noncollagenolytic mechanisms, MMP-1 silencing in HEp3-hi/diss cells modulated the microarchitecture and integrity of the angiogenic vasculature in a novel microtumor model. Concomitantly, MMP-1 deficiency led to decreased levels of intratumoral vascular permeability, tumor cell intravasation, and metastatic dissemination. Taking advantage of PAR1 deficiency of HEp3-hi/diss cells, we further show that endothelial PAR1 is a putative nontumor-cell/nonmatrix target, activation of which by carcinoma-produced MMP-1 regulates endothelial permeability and transendothelial migration. The inhibitory effects of specific PAR1 antagonists in live animals have also indicated that the mechanisms of MMP-1-dependent vascular permeability in tumors involve endothelial PAR1 activation. Together, our findings mechanistically underscore the contribution of a tumor MMP-1/endothelial PAR1 axis to actual intravasation events manifested by aggressive carcinoma cells.

Neuropilin-2 Promotes Extravasation and Metastasis by Interacting with Endothelial α5 Integrin

Metastasis, the leading cause of cancer death, requires tumor cell intravasation, migration through the bloodstream, arrest within capillaries, and extravasation to invade distant tissues. Few mechanistic details have been reported thus far regarding the extravasation process or re-entry of circulating tumor cells at metastatic sites. Here, we show that neuropilin-2 (NRP-2), a multifunctional nonkinase receptor for semaphorins, vascular endothelial growth factor (VEGF), and other growth factors, expressed on cancer cells interacts with α5 integrin on endothelial cells to mediate vascular extravasation and metastasis in zebrafish and murine xenograft models of clear cell renal cell carcinoma (RCC) and pancreatic adenocarcinoma. In tissue from patients with RCC, NRP-2 expression is positively correlated with tumor grade and is highest in metastatic tumors. In a prospectively acquired cohort of patients with pancreatic cancer, high NRP-2 expression cosegregated with poor prognosis. Through biochemical approaches as well as Atomic Force Microscopy (AFM), we describe a unique mechanism through which NRP-2 expressed on cancer cells interacts with α5 integrin on endothelial cells to mediate vascular adhesion and extravasation. Taken together, our studies reveal a clinically significant role of NRP-2 in cancer cell extravasation and promotion of metastasis.

C-Jun N-Terminal Kinases Mediate a Wide Range of Targets in the Metastatic Cascade

Disseminated cancer cells rely on intricate interactions among diverse cell types in the tumor-associated stroma, vasculature, and immune system for survival and growth. Ubiquitous expression of c-Jun N-terminal kinase (jnk) genes in various cell types permits their control of metastasis. In early stages of metastasis, JNKs affect tumor-associated inflammation and angiogenesis as well as tumor cell migration and intravasation. Within the tumor stroma, JNKs are essential for the release of growth factors that promote epithelial-to-mesenchymal transition (EMT) in tumor cells. JNK3, the least ubiquitous isoform, facilitates angiogenesis by increasing endothelial cell migration. Importantly, JNK expression in tumor cells integrates stromal signals to promote tumor cell invasion. However, JNK isoforms differentially regulate migration toward the endothelial barrier. Once tumor cells enter the bloodstream, JNKs increase circulating tumor cell (CTC) survival and homing to tissues. By promoting fibrosis, JNKs improve CTC attachment to the endothelium. Once anchored, JNKs stimulate EMT to facilitate tumor cell extravasation and enhance the secretion of endothelial barrier disrupters. Tumor cells attract barrier-disrupting macrophages by JNK-dependent transcription of macrophage chemoattractant molecules. In the secondary tissue, JNKs are instrumental in the premetastatic niche and stimulate tumor cell proliferation. JNK expression in cancer cells stimulates tissue-remodeling macrophages to improve tumor colonization. However, in T-cells, JNKs alter cytokine production that increases tumor surveillance and inhibits the recruitment of tissue-remodeling macrophages. Therapeutically targeting JNKs for metastatic disease is attractive considering their promotion of metastasis; however, specific JNK tools are needed to determine their definitive actions within the context of the entire metastatic cascade.

Abstract 2685: Regulation of melanoma metastasis by malignant melanoma-initiating cell marker ABCB5.

Abstract Cancer stem cells drive tumor growth, including metastatic disease progression. ATP-binding cassette member B5 (ABCB5) expression identifies malignant melanoma initiating cells (MMICs), which are distinguishable from cancer bulk populations by their self-renewal and differentiation capacities, and pro-oncogenic interactions with the microenvironment. ABCB5 confers drug resistance in multiple human malignancies through its actions as a drug efflux transporter. Here, we demonstrate that ABCB5 also regulates melanoma metastasis. ABCB5 monoclonal antibody (mAb) treatment significantly inhibited metastasis development in highly immune-compromised NOD-scid IL2Rγnull mice carrying fully established human melanoma xenografts, independent of previously reported antibody-dependent cell-mediated cytotoxicity (ADCC) effects. ABCB5 mAb treatment was well tolerated, as indicated by equal body weights of treated mice and untreated controls. Moreover, ABCB5 mAb also inhibited melanoma cell invasive capacity, suggesting that ABCB5 regulates intravasation and/or extravasation of metastatic tumor cells. Furthermore, the average size of metastatic nodules in ABCB5 mAb-treated mice was significantly smaller than those in isotype control mAb-treated or untreated mice, indicating that ABCB5 also regulates proliferation of disseminated tumor cells. Expression profile analysis revealed that multiple known metastasis-associated genes are regulated by ABCB5. Rescue of their expression could reverse the anti-metastatic effects induced by ABCB5 inhibition. Thus, ABCB5 is not only a marker for MMICs, but functionally drives metastatic melanoma progression. Citation Format: Jie Ma, Allireza Alloo, Brian J. Wilson, Markus H. Frank. Regulation of melanoma metastasis by malignant melanoma-initiating cell marker ABCB5. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2685. doi:10.1158/1538-7445.AM2013-2685 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 2301: Myeloid cells promote fibroblast invasion and lead mammary adenocarcinoma cell invasion.

Abstract Myeloid cells localize to invasive edges of solid tumors where they promote invasive tumor growth, intravasation into vasculature and metastasis of malignant cells1,2. Fibroblasts, on the other hand, lead collective cell migration3 of carcinoma cells via fostering extracellular matrix (ECM) remodeling and generating tracks of collagen fibers that act as “highways” for cell migration4,5. To identify soluble molecules mediating these juxtacrine communication mechanisms between myeloid cells and fibroblasts in potentiating metastasis, we conducted pilot studies with subsets of spleen- or tumor-derived myeloid cells, tumor-derived fibroblasts, and mammary adenocarcinoma cells. Whereas spleen-derived CD11b+Ly6ChiLy6G− monocytes promote fibroblast migration ex vivo, two populations of tumor-derived myeloid cells, e.g., CD11b+Gr1+ and CD11b+Gr1− cells possessed this capability. In ex vivo migration assays, fibroblast migration was dependent on myeloid cell-derived factors, e.g., CXCL11, CXCL15, FGF2, IGF-1 and Shh, as well as fibroblast Akt and ERK1/2 phosphorylation. Inhibition of fibroblast MEK reduced myeloid cell-induced fibroblast migration, whereas FGF-RI inhibition completely abolished this capability. These studies indicate a role for myeloid cell-fibroblast communication in fostering migration (and potentially activation) of fibroblasts that lead collective cell migration properties of mammary carcinoma cells. Ongoing studies are identifying specific subpopulations of tumor-associated myeloid cells possessing these activities, as well as the role of FGF receptor signaling pathways in facilitating metastasis. 1. Wyckoff, J.B., et al. Direct visualization of macrophage-assisted tumor cell intravasation in mammary tumors. Cancer Res 67, 2649-2656 (2007). 2. Qian, B.Z. & Pollard, J.W. Macrophage diversity enhances tumor progression and metastasis. Cell 141, 39-51 (2010). 3. Friedl, P., Locker, J., Sahai, E. & Segall, J.E. Classifying collective cancer cell invasion. Nat Cell Biol 14, 777-783 (2012). 4. Khalil, A.A. & Friedl, P. Determinants of leader cells in collective cell migration. Integr Biol (Camb) 2, 568-574 (2010). 5. Gritsenko, P.G., Ilina, O. & Friedl, P. Interstitial guidance of cancer invasion. J Pathol 226, 185-199 (2012). The authors acknowledge generous support from the NIH/NCI, a Dept of Defense (DoD) Breast Cancer Research Program Postdoctoral Fellowship, a DoD Era of Hope Scholar Expansion Award, Susan G. Komen Fndt, and the Breast Cancer Research Fndt. Citation Format: Aubie K. Shaw, Sergey Novitskiy, Lisa M. Coussens, Harold L. Moses. Myeloid cells promote fibroblast invasion and lead mammary adenocarcinoma cell invasion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2301. doi:10.1158/1538-7445.AM2013-2301

In vitro characterisation of the anti-intravasative properties of the marine product heteronemin

Metastases destroy the function of infested organs and are the main reason of cancer-related mortality. Heteronemin, a natural product derived from a marine sponge, was tested in vitro regarding its properties to prevent tumour cell intravasation through the lymph-endothelial barrier. In three-dimensional (3D) cell cultures consisting of MCF-7 breast cancer cell spheroids that were placed on lymph-endothelial cell (LEC) monolayers, tumour cell spheroids induce "circular chemorepellent-induced defects" (CCIDs) in the LEC monolayer; 12(S)-Hydroxyeicosatetraenoic acid (12(S)-HETE) and NF-κB activity are major factors inducing CCIDs, which are entry gates for tumour emboli intravasating the vasculature. This 3D co-culture is a validated model for the investigation of intravasation mechanisms and of drugs preventing CCID formation and hence lymph node metastasis. Furthermore, Western blot analyses, NF-κB reporter, EROD, SELE, 12(S)-HETE, and adhesion assays were performed to investigate the properties of heteronemin. Five micromolar heteronemin inhibited the directional movement of LECs and, therefore, the formation of CCIDs, which were induced by MCF-7 spheroids. Furthermore, heteronemin reduced the adhesion of MCF-7 cells to LECs and suppressed 12(S)-HETE-induced expression of the EMT marker paxillin, which is a regulator of directional cell migration. The activity of CYP1A1, which contributed to CCID formation, was also inhibited by heteronemin. Hence, heteronemin inhibits important mechanisms contributing to tumour intravasation in vitro and should be tested in vivo.

STC1 Expression By Cancer-Associated Fibroblasts Drives Metastasis of Colorectal Cancer

Platelet-derived growth factor (PDGF) receptor signaling is a major functional determinant of cancer-associated fibroblasts (CAF). Elevated expression of PDGF receptors on stromal CAFs is associated with metastasis and poor prognosis, but mechanism(s) that underlie these connections are not understood. Here, we report the identification of the secreted glycoprotein stanniocalcin-1 (STC1) as a mediator of metastasis by PDGF receptor function in the setting of colorectal cancer. PDGF-stimulated fibroblasts increased migration and invasion of cocultured colorectal cancer cells in an STC1-dependent manner. Analyses of human colorectal cancers revealed significant associations between stromal PDGF receptor and STC1 expression. In an orthotopic mouse model of colorectal cancer, tumors formed in the presence of STC1-deficient fibroblasts displayed reduced intravasation of tumor cells along with fewer and smaller distant metastases formed. Our results reveal a mechanistic basis for understanding the contribution of PDGF-activated CAFs to cancer metastasis.

Abstract A84: Synaptojanin-2 and microRNA-31 control invadopodia and metastasis by regulating vesicular trafficking

Abstract Growth factors propel cell migration and metastasis, but underlying mechanisms are incompletely understood. Employing EGF-stimulated mammary cells we link the lipid phosphatase synaptojanin 2 (SYNJ2) to an invasive phenotype, and relate high SYNJ2 and low microRNA-31, a metastasis suppressor that restrains SYNJ2, to short survival rates of cancer patients. Knockdown of SYNJ2 robustly impaired both intravasation and extravasation of mammary tumor cells in an animal model. In vitro, SYNJ2-depleted cells exhibited derailed trafficking of EGFR and integrins, resulting in deformed focal adhesions, arrested lamellipodia and disappearance of invadopodia. We conclude that recycling of active EGFRs focally promotes SYNJ2-mediated dephosphorylation of specific phosphoinositol lipids, thereby instigates formation of both invadopodia and lamellipodia and facilitates tumor progression. Citation Format: Nir Ben-Chetrit, David Chetrit, Cindy Koerner, Silvia Carvalho, Marc Symons, Fernando Schmitt, Stefan Wiemann, Marcelo Ehrlich, Yosef Yarden. Synaptojanin-2 and microRNA-31 control invadopodia and metastasis by regulating vesicular trafficking. [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 A84.

Abstract C18: Matrix metalloproteinase-1 (MMP-1) facilitates intravasation and vascular dissemination of human carcinoma cells by regulating the microarchitecture and permeability of intratumoral vasculature

Abstract Patients with squamous cell carcinomas (SCCs) often present with advanced stage disease and face one of the lowest 5-year post-therapeutic survival rates. Among 25 signature genes, Mmp1 has been found to be third best SCC tumor predictor, suggesting a critical role of MMP-1 in SCC progression. Functional role of MMP-1 in SCCs was analyzed in this study by employing a highly-disseminating variant of human epidermoid HEp-3 carcinoma, HEp3-hi/diss. The dissemination capacity of HEp3-hi/diss cells in chick embryo spontaneous metastasis models positively correlated with their high levels of MMP-1 expression. MMP-1-deficiency induced by RNA interference resulted in 70-95% inhibition of HEp3-hi/diss intravasation, concomitant with collapse of the intravasation-sustaining microarchitecture of intratumoral vasculature. Furthermore, we have demonstrated the mechanism whereby HEp3-hi/diss-produced MMP-1 contributes to tumor cell intravasation by proteolytically inducing vascular endothelial permeability and increasing rates of carcinoma cells crossing over vascular barriers during the intravasation step of the metastatic cascade. Citation Format: Anna Juncker-Jensen, Berta Casar, Ivo Rimann, Ewa Zajac, Tatyana A. Kupriyanova, Elena I. Deryugina, James P. Quigley. Matrix metalloproteinase-1 (MMP-1) facilitates intravasation and vascular dissemination of human carcinoma cells by regulating the microarchitecture and permeability of intratumoral vasculature. [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 C18.

Abstract C26: Molecular determinants of the intravasation step in cancer metastasis

Abstract Intravasation, the directional entry of primary tumor cells into host vasculature, remains the most understudied step in the metastatic cascade, likely due to deficiency in models recapitulating the intravasation process and allowing for molecular probing and intervention in vivo. By employing chick embryo models of spontaneous metastasis, where human tumor cells form microtumors and disseminate into secondary tissues strictly via an angiogenic vasculature, we have identified several rate-limiting processes and critical molecules of tumor and host origin, which functionally contribute to intravasation. We have demonstrated that the induction of an intravasation-sustaining tumor microenvironment involves tumor-produced cytokines, including IL-8 that triggers the influx of neutrophils into developing primary tumors. These inflammatory neutrophils deliver a potent pro-angiogenic protease, TIMP-1-free MMP-9, which releases stroma-bound angiogenic factors such as FGF-2 and VEGF. In addition, our recent findings indicate that M2-polarized macrophages also produce TIMP-1-deficient MMP-9, which explains the high angiogenic potential of this sub-type of inflammatory cells. Whereas host inflammatory cell MMP-9 promotes tumor angiogenesis, one of the tumor-produced MMPs, namely MMP-1, appears to regulate the microarchitecture of intratumoral vasculature. We have demonstrated that carcinoma cell MMP-1 facilitates tumor cell intravasation by specific induction of vascular permeability via activation of PAR1 expressed on the angiogenic endothelial cells. Finally, we show that intimate interactions between tumor cells and host endothelial cells result in de novo generation of plasmin that cleaves a transmembrane molecule, CDCP1, regulating tumor cell survival and motility signaling, which allows for successful intravasation. Together, our findings illuminate how different proteolytic enzymes, implicated mostly in path-clearing matrix proteolysis and tumor invasion, also orchestrate localized protein modifications and regulate cellular processes leading to, and following the intratumoral intravasation events. Citation Format: Elena I. Deryugina, James P. Quigley. Molecular determinants of the intravasation step in cancer 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 C26.

Abstract ES8-1: Inflammation and Breast Cancer

Abstract Mechanism-based insights into prognosis and treatment of breast cancer derived from high resolution multiphoton imaging John Condeelis, Thomas Rohan, Maja Oktay - Einstein, New York; Tony Ng – King's College London, UK; Seema Agarwal, David Rimm – Yale, New Haven; Brian Robinson, Joan Jones – Weill Cornell Medical Center, New York Frank Gertler MIT, Boston. Multi-photon microscopy allows the observation of tumor cell behavior at single cell resolution in vivo and has demonstrated that invasive and migratory tumor cells in mammary tumors form migratory streams and intravasate only when associated with macrophages. This analysis demonstrates that tumor cell streaming and intravasation are cell autonomous behaviors that are the direct result of the tumor cell -macrophage interaction. The in vivo invasion assay was developed to collect these streaming and intravasation competent tumor cells from primary tumors in vivo. Collection of these cells was coupled to expression profiling to reveal the genes expressed by tumor cells during streaming and intravasation in rat, mouse and human breast tumors (the Invasion Signature). The invasion, adhesion and motility pathways identified in the Invasion Signature converge on the RhoC/Cofilin/Mena pathway identifying it as a master regulator of chemotaxis, streaming and intravasation of breast tumor cells in vivo. Using prognostic markers derived from the RhoC/Cofilin/Mena pathway, anatomical landmarks have been developed for use with breast cancer patients. One of these, composed of the tumor cell-macrophage intravasation complex, is called TMEM (Tumor MicroEnvironment for Metastasis) in human breast tumors. TMEM density in histology sections of breast tumor tissue has been found to predict metastatic risk. The potential for tumor cell/macrophage pairing, steaming and TMEM assembly can be detected in breast tumors by the relative expression of isoforms of Mena-INV (enhancer) and Mena11a (suppressor of migration and dissemination, referred to as the MenaCalc test). Two retrospective studies with breast cancer tissue from patients with known recurrence and survival outcome have demonstrated the use, for prediction of metastatic risk, of the MenaCalc test and the TMEM test in resected breast tumor tissue. A CofilinxP-Cofilin test, also derived from the RhoC/Cofilin/Mena pathway of the human Invasion Signature, is showing promise as a predictor of disease-free survival. The Cofilin part of the RhoC/Mena/Cofilin pathway is associated with the overall activity of a biologically relevant sub-network of proteins (centered around four seed proteins ezrin/radixin/moesin/cofilin) in the cancer proteome that control membrane-cytoskeletal linkage and tumor cell motility. CofilinxP-Cofilin co-localization intensity was the most biologically significant variable measured among these others in predicting survival in a large retrospective trial of breast cancer patients. The value of this test derives from the fact that it is independent of anti-estrogen (hormonal) treatment and chemotherapy. In summary, multi-photon imaging in deep tissue locations in live animals in real time has revealed molecular mechanisms associated with complex cancer cell behavior during metastatic dissemination leading directly to new markers of metastatic risk and survival. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr ES8-1.

In vivo cleaved CDCP1 promotes early tumor dissemination via complexing with activated β1 integrin and induction of FAK/PI3K/Akt motility signaling

Specific cleavage of the transmembrane molecule, CUB domain-containing protein-1 (CDCP1), by plasmin-like serine proteases induces outside-in signal transduction that facilitates early stages of spontaneous metastasis leading to tumor cell intravasation, namely cell escape from the primary tumor, stromal invasion and transendothelial migration. We identified active β1 integrin as a biochemical and functional partner of the membrane-retained 70-kDa CDCP1 fragment, newly generated from its full-length 135-kDa precursor though proteolytic cleavage by serine proteases. Both in cell cultures and in live animals, active β1 integrin complexed preferentially with functionally activated, phosphorylated 70-kDa CDCP1. Complexing of β1 integrin the 70-kDa with CDCP1 fragment induced intracellular phosphorylation signaling, involving focal adhesion kinase-1 (FAK) and PI3 kinase (PI3K)-dependent Akt activation. Thus, inhibition of FAK/PI3K activities by specific inhibitors as well as short-hairpin RNA downregulation of β1 integrin significantly reduced FAK/Akt phosphorylation under conditions where CDCP1 was processed by serine proteases, indicating that FAK/PI3K/Akt pathway operates downstream of cleaved CDCP1 complexed with β1 integrin. Furthermore, this complex-dependent signaling correlated positively with high levels of tumor cell intravasation and dissemination. Correspondingly, abrogation in vivo of CDCP1 cleavage either by unique cleavage-blocking monoclonal antibody 10-D7 or by inhibition of proteolytic activity of plasmin-like serine proteases with aprotinin prevented β1 integrin/CDCP1 complexing and downstream FAK/Akt signaling concomitant with significant reduction of stromal invasion and spontaneous metastasis. Therefore, β1 integrin appears to serve as a motility-regulating partner mediating cross-talk between proteolytically cleaved, membrane-retained CDCP1 and members of FAK/PI3K/Akt pathway. This CDCP1 cleavage-induced signaling cascade constitutes a unique mechanism, independent of extracellular matrix remodeling, whereby a proteolytically cleaved CDCP1 regulates in vivo locomotion and metastasis of tumor cells through β1 integrin partnering. Our findings indicate that CDCP1 cleavage, occurring at the apex of a β1 integrin/FAK/PI3K/Akt signaling cascade, may represent a therapeutic target for CDCP1-positive cancers.

Targeting Tumor Cell Invasion and Dissemination In Vivo by an Aptamer That Inhibits Urokinase-type Plasminogen Activator through a Novel Multifunctional Mechanism

Data accumulated over the latest two decades have established that the serine protease urokinase-type plasminogen activator (uPA) is a potential therapeutic target in cancer. When designing inhibitors of the proteolytic activity of serine proteases, obtaining sufficient specificity is problematic, because the topology of the proteases' active sites are highly similar. In an effort to generate highly specific uPA inhibitors with new inhibitory modalities, we isolated uPA-binding RNA aptamers by screening a library of 35 nucleotides long 2'-fluoro-pyrimidine RNA molecules using a version of human pro-uPA lacking the epidermal growth factor-like and kringle domains as bait. One pro-uPA-binding aptamer sequence, referred to as upanap-126, proved to be highly specific for human uPA. Upanap-126 delayed the proteolytic conversion of human pro-uPA to active uPA, but did not inhibit plasminogen activation catalyzed by two-chain uPA. The aptamer also inhibited the binding of pro-uPA to uPAR and the binding of vitronectin to the preformed pro-uPA/uPAR complex, both in cell-free systems and on cell surfaces. Furthermore, upanap-126 inhibited human tumor cell invasion in vitro in the Matrigel assay and in vivo in the chick embryo assay of cell escape from microtumors. Finally, upanap-126 significantly reduced the levels of tumor cell intravasation and dissemination in the chick embryo model of spontaneous metastasis. Together, our findings show that usage of upanap-126 represents a novel multifunctional mechanistic modality for inhibition of uPA-dependent processes involved in tumor cell spread.

Bay11-7082 inhibits the disintegration of the lymphendothelial barrier triggered by MCF-7 breast cancer spheroids; the role of ICAM-1 and adhesion

Background:Many cancers spread through lymphatic routes, and mechanistic insights of tumour intravasation into the lymphatic vasculature and targets for intervention are limited. The major emphasis of research focuses currently on the molecular biology of tumour cells, while still little is known regarding the contribution of lymphatics.Methods:Breast cancer cell spheroids attached to lymphendothelial cell (LEC) monolayers were used to investigate the process of intravasation by measuring the areas of ‘circular chemorepellent-induced defects' (CCID), which can be considered as entry gates for bulky tumour intravasation. Aspects of tumour cell intravasation were furthermore studied by adhesion assay, and siRNA-mediated knockdown of intracellular adhesion molecule-1 (ICAM-1). Replacing cancer spheroids with the CCID-triggering compound 12(S)-hydroxyeicosatetraenoic acid (HETE) facilitated western blot analyses of Bay11-7082- and baicalein-treated LECs.Results:Binding of LECs to MCF-7 spheroids, which is a prerequisite for CCID formation, was mediated by ICAM-1 expression, and this depended on NF-κB and correlated with the expression of the prometastatic factor S100A4. Simultaneous inhibition of NF-κB with Bay11-7082 and of arachidonate lipoxygenase (ALOX)-15 with baicalein prevented CCID formation additively.Conclusion:Two mechanisms contribute to CCID formation: ALOX15 via the generation of 12(S)-HETE by MCF-7 cells, which induces directional migration of LECs, and ICAM-1 in LECs under control of NF-κB, which facilitates adhesion of MCF-7 cells to LECs.