Jennifer M. Roth

Thrombin Up-regulates Cathepsin D which Enhances Angiogenesis, Growth, and Metastasis

Cathepsin D (CD) up-regulation has been associated with human malignancy and poor prognosis. Thrombin up-regulated CD mRNA and protein in eight tumor cell lines as well as in human umbilical vascular endothelial cells (HUVEC). Thrombin increased the secretion of CD by 3- to 8-fold and enhanced chemotaxis ( approximately 2-fold) in 4T1 murine mammary CA cells, which was completely inhibited with the knockdown of CD. Secreted 4T1 CD induced neoangiogenesis by 2.4-fold on a chick chorioallantoic membrane, which was blocked in CD-KD cells. The addition of pure CD (2 ng) to the chick chorioallantoic membrane increased angiogenesis by 2.1-fold, which was completely inhibited by Pepstatin A (Pep A). CD enhanced human HUVEC chemotaxis and Matrigel tube formation by 2-fold, which was then blocked by Pep A. CD enhanced HUVEC matrix metalloproteinase 9 (MMP-9) activity by approximately 2-fold, which was completely inhibited by Pep A as well as a generic MMP inhibitor, GM6001. The injection of CD-KD 4T1 cells into syngeneic mice inhibited tumor growth by 3- to 4-fold compared with empty vector (EV) cells. Hirudin, a specific thrombin inhibitor, inhibited the growth of wild-type and EV cells by 2- to 3-fold, compatible with thrombin up-regulation of CD. CD and thrombin also contributed to spontaneous pulmonary metastasis; 4-fold nodule inhibition with CD versus EV and 4.6-fold inhibition with hirudin versus EV (P < 0.02). Thus, thrombin-induced CD contributes to the malignant phenotype by inducing tumor cell migration, nodule growth, metastasis, and angiogenesis. CD-induced angiogenesis requires the proteolytic activation of MMP-9.

Twist Is Required for Thrombin-Induced Tumor Angiogenesis and Growth

Twist, a master regulator of embryonic morphogenesis, induces functions that are also required for tumor invasion and metastasis. Because thrombin contributes to the malignant phenotype by up-regulating tumor metastasis, we examined its effect on Twist in five different tumor cell lines and two different endothelial cell lines. Thrombin up-regulated Twist mRNA and protein in all seven cell lines. Down-regulation of Twist in B16F10 tumor cell lines led to a approximately 3-fold decrease in tumor growth on a chorioallantoic membrane assay and approximately 2-fold decrease in syngeneic mice. Angiogenesis was decreased approximately 45% and 36%, respectively. The effect of Twist on angiogenesis was further examined and compared with the effect of thrombin. In studies using a Twist-inducible plasmid, several identical vascular growth factors and receptors were up-regulated approximately 2- to 3-fold in tumor cells as well as human umbilical vascular endothelial cells by both Twist as well as thrombin (vascular endothelial growth factor, KDR, Ang-2, matrix metalloproteinase 1, GRO-alpha, and CD31). Thrombin-induced endothelial cell chemotaxis and Matrigel endothelial cell tubule formation were similarly regulated by Twist. Thus, thrombin up-regulates Twist, which is required for thrombin-induced angiogenesis as measured by endothelial cell migration, Matrigel tubule formation, and tumor angiogenesis.

The Cochaperone p23 Differentially Regulates Estrogen Receptor Target Genes and Promotes Tumor Cell Adhesion and Invasion

ABSTRACT The cochaperone p23 plays an important role in estrogen receptor alpha (ER) signal transduction. In this study, we investigated how p23 regulates ER target gene activation and affects tumor growth and progression. Remarkably, we found that changes in the expression of p23 differentially affected the activation of ER target genes in a manner dependent upon the type of DNA regulatory element. p23 overexpression enhanced the expression of the ER target genes cathepsin D and pS2, which are regulated by direct DNA binding of ER to estrogen response elements (ERE). In contrast, the expression of other target genes, including c-Myc, cyclin D1, and E2F1, to which ER is recruited indirectly through its interaction with other transcription factors remains unaffected by changes in p23 levels. The p23-induced expression of pS2 is associated with enhanced recruitment of ER to the ERE in the promoter, whereas ER recruitment to the ERE-less c-Myc promoter does not respond to p23. Intriguingly, p23-overexpressing MCF-7 cells exhibit increased adhesion and invasion in the presence of fibronectin. Our findings demonstrate that p23 differentially regulates ER target genes and is involved in the control of distinct cellular processes in breast tumor development, thus revealing novel functions of this cochaperone.

Recombinant α2(IV)NC1 Domain Inhibits Tumor Cell-Extracellular Matrix Interactions, Induces Cellular Senescence, and Inhibits Tumor Growth in Vivo

Cellular interaction with the extracellular matrix is thought to be a critical event in controlling angiogenesis and tumor growth. In our previous studies, genetically distinct noncollagenous (NC) domains of type-IV collagen were shown to interact with integrin receptors expressed on the surface of endothelial cells. Moreover, these NC1 domains were shown to inhibit angiogenesis in vivo. Here, we provide evidence that a recombinant form of the α2(IV)NC1 domain of type-IV collagen could bind integrins α1β1 and αvβ3 expressed on melanoma cells and inhibit tumor cell adhesion in a ligand-specific manner. Systemic administration of recombinant α2(IV)NC1 domain potently inhibited M21 melanoma tumor growth within full thickness human skin and exhibited a dose-dependent inhibition of tumor growth in nude mice. Interestingly, α2(IV)NC1 domain enhanced cellular senescence in tumor cells in vitro and in vivo. Taken together, these results suggest that recombinant α2(IV)NC1 domain is not only a potent anti-angiogenic reagent, but it also directly impacts tumor cell behavior. Thus, α2(IV)NC1 domain represents a potent inhibitor of tumor growth by impacting both endothelial and tumor cell compartments.

Disruption of Endothelial Cell Interactions with the Novel HU177 Cryptic Collagen Epitope Inhibits Angiogenesis

Purpose: The importance of cellular communication with the extracellular matrix in regulating cellular invasion is well established. Selective disruption of communication links between cells and the local microenvironment by specifically targeting non-cellular matrix-immobilized cryptic extracellular matrix epitopes may represent an effective new clinical approach to limit tumor-associated angiogenesis. Therefore, we sought to determine whether the HU177 cryptic collagen epitope plays a functional role in regulating angiogenesis in vivo. Experimental Design: We examined the expression and characterized the HU177 cryptic collagen epitope in vitro and in vivo using immunohistochemistry and ELISA. We examined potential mechanisms by which this cryptic collagen epitope may regulate angiogenesis using in vitro cell adhesion, migration, proliferation, and biochemical assays. Finally, we examined the whether blocking cellular interactions with the HU177 cryptic epitope plays a role in angiogenesis and tumor growth in vivo using the chick embryo model. Results: The HU177 cryptic epitope was selectively exposed within tumor blood vessel extracellular matrix, whereas little was associated with quiescent vessels. An antibody directed to this cryptic site selectively inhibited endothelial cell adhesion, migration, and proliferation on denatured collagen type IV and induced increased levels of cyclin-dependent kinase inhibitor p27KIP1. Systemic administration of mAb HU177 inhibited cytokine- and tumor-induced angiogenesis in vivo. Conclusions: We provide evidence for a new functional cryptic regulatory element within collagen IV that regulates tumor angiogenesis. These findings suggest a novel and highly selective approach for regulating angiogenesis by targeting a non-cellular cryptic collagen epitope.

Inhibition of Angiogenesis and Tumor Metastasis by Targeting a Matrix Immobilized Cryptic Extracellular Matrix Epitope in Laminin

Angiogenesis and tumor metastasis depend on extracellular matrix (ECM) remodeling and subsequent cellular interactions with these modified proteins. An in-depth understanding of how both endothelial and tumor cells use matrix-immobilized cryptic ECM epitopes to regulate invasive cell behavior may lead to the development of novel strategies for the treatment of human tumors. However, little is known concerning the existence and the functional significance of cryptic laminin epitopes in regulating angiogenesis and tumor cell metastasis. Here, we report the isolation and characterization of a synthetic peptide that binds to a cryptic epitope in laminin. The STQ peptide selectively bound denatured and proteolyzed laminin but showed little interaction with native laminin. The cryptic laminin epitope recognized by this peptide was selectively exposed within malignant melanoma in vivo, whereas little if any was detected in normal mouse skin. Moreover, the STQ peptide selectively inhibited endothelial and tumor cell adhesion, migration, and proliferation in vitro and inhibited angiogenesis, tumor growth, and experimental metastasis in vivo. This inhibitory activity was associated with a selective up-regulation of the cyclin-dependent kinase inhibitor P27(KIP1) and induction of cellular senescence. These novel findings suggest the existence of functionally relevant cryptic laminin epitopes in vivo and that selective targeting of these laminin epitopes may represent an effective new strategy for the treatment of malignant tumors by affecting both the endothelial and tumor cell compartments.

Ionizing radiation modulates the exposure of the HUIV26 cryptic epitope within collagen type IV during angiogenesis

PURPOSE The majority of the research on the biologic effects of ionizing radiation has focused on the impact of radiation on cells in terms of gene expression, DNA damage, and cytotoxicity. In comparison, little information is available concerning the direct effects of radiation on the extracellular microenvironment, specifically the extracellular matrix and its main component, collagen. We have developed a series of monoclonal antibodies that bind to cryptic epitopes of collagen Type IV that are differentially exposed during matrix remodeling and are key mediators of angiogenesis. We have hypothesized that ionizing radiation might affect the process of angiogenesis through a direct effect on the extracellular matrix and specifically on collagen Type IV. METHODS AND MATERIALS Angiogenesis was induced in a chick chorioallantoic membrane (CAM) model; 24 h later, a single-dose treatment with ionizing radiation (0.5, 5, and 20 cGy) was administered. Angiogenesis was assessed, and the exposure of two cryptic regulatory epitopes within collagen Type IV (HUI77 and HUIV26) was studied in vitro by solid-phase ELISA and in vivo by immunofluorescence staining. RESULTS A dose-dependent reduction of angiogenesis with maximum inhibition (85%-90%) occurring at 20 cGy was demonstrated in the CAM model. Exposure of the cryptic HUIV26 site, an angiogenesis control element, was inhibited both in vitro and in vivo by the same radiation dose, whereas little if any change was observed for the HUI77 cryptic epitope. CONCLUSIONS A dose-dependent alteration of the functional exposure of the HUIV26 cryptic epitope is induced by radiation in vitro and in the CAM model in vivo. This radiation-induced change in protein structure and function may contribute to the inhibitory effects of ionizing radiation on new blood vessel growth and warrants further studies in other models.

Cooperative Interactions Between Integrins and Growth Factor Signaling in Pathological Angiogenesis

As with most complex biological processes, angiogenesis requires the integration of a number of molecular signaling networks to coordinate multiple cues from both the extracellular tissue microenvironment as well as the cell’s interior. Thus an important area of angiogenesis investigation involves understanding the mechanisms that facilitate cooperation between multiple receptor–ligand signaling pathways. Two crucial networks that play active role in angiogenesis include growth factor/growth factor receptors and extracellular matrix/integrin receptor signaling systems. Emerging evidence suggests that these two important signaling systems depend in large part on each other, and function cooperatively to control new blood vessel development. Given the tissue-specific variations in the expression of components within each of these systems, significant challenges exist in order to exploit these signaling pathways for clinical intervention. A more detailed understanding of how the molecular components of these two signaling systems communicate with each other to direct and coordinate downstream effector functions may lead to optimized anti-angiogenic strategies to control malignant tumor progression. In this regard, we will discuss the multiple ways by which growth factor and integrin signaling pathways function cooperatively to regulate pathological angiogenesis within the context of the tissue microenvironment.

Abstract 4025: Accelerating drug repurposing for cancer therapy using multiplexed viability assays

New therapies are desperately needed for patients with advanced cancer, but making safe and efficacious drugs remains expensive and time consuming. Meanwhile, multiple non-oncology drugs have been successfully repurposed for cancer, including thalidomide for multiple myeloma and aspirin for prevention of colorectal cancer. While the benefits of repurposing are clear, successes to date have been largely serendipitous. We have created two foundational tools to identify drug repurposing opportunities at a much greater scale: a new world-class screening library of more than 4,000 drugs/clinical candidates and a multiplexed method to rapidly conduct cellular viability assays across hundreds of cell lines. PRISM, a recently developed high-throughput assay, employs DNA-barcoded cell lines to enable rapid testing of many drugs against pools of cancer cell lines. We have tested 4,100 compounds against 578 genomically characterized cancer cell lines using PRISM. Our experiment is among the largest cell line screens ever undertaken. Global analysis has revealed multiple strong clusters of active drugs, including vitamin D agonists, bromodomain inhibitors, and statins. In addition, many novel and established drug-biomarker relationships were identified, including alkylating agent response and low MGMT expression, BRAF inhibitor response and BRAF mutation, and MDM2 inhibitor response and TP53 wild-type status. Surprisingly, more than 100 non-oncology drugs unexpectedly killed multiple cancer cell lines, making them attractive repurposing candidates. For example, the FDA-approved phosphodiesterase inhibitor anagrelide selectively kills PDE3A-high lung cancer and melanoma cell lines. Confirmatory experiments are underway to validate screening results, evaluate putative biomarkers, and test drug activity in preclinical models. Importantly, this approach is expected to enable rapid initiation of clinical trials, dramatically accelerating patient access to potential new therapies. Citation Format: Steven M. Corsello, Christopher C. Mader, Jordan Bryan, Jennifer Roth, David Peck, John Davis, Samantha Bender, Li Wang, Alice Wang, Joshua Bittker, Francisca Vazquez, Aravind Subramanian, Aviad Tsherniak, Todd R. Golub. Accelerating drug repurposing for cancer therapy using multiplexed viability assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4025. doi:10.1158/1538-7445.AM2017-4025

Abstract 4617: Hu177 cryptic epitope: A novel biomarker for the monitoring of treatment of ovarian cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Distinct cryptic collagen epitopes are among the protein fragments exposed by collagen type IV remodeling, and recent data indicate that these cryptic epitopes may facilitate tumor migration and angiogenesis. In studies with melanoma patients we tested the hypothesis that melanoma can induce detectable changes in systemic levels of cryptic epitope shedding, specifically the HU177 epitope and were able correlate the levels of HU177 shedding with clinical and pathologic parameters (Ng et al Clin Can Res, 14:6253,2008). In addition in a follow-up study the increased shedding of the Hu177 epitope was shown to correlate with a worse prognosis in primary melanoma (Hamilton et al, J Trans Med8:19,2010). In this study we examined wether the serum levels of these unique cryptic epitopes may be useful to monitor, in a phase II study with ovarian patients, the course of the combination treatment of continuous infusion topotecan with erlotinib over multiple cycles of therapeutic intervention. Methods: We made use of our previously published ELISA serum assay (Ng et al) of which the key components involve the anchoring of the primary Hu177 directed antibody followed by blocking with albumin and then incubation with patient serum. The assay is further developed with a sandwich ELISA consisting of biotinylated anti-collagen IV and an anti-biotin antibody conjugated to horseradish peroxidase, followed by chromagenic color development. A parallel examination of the Hu177 epitope was also conducted with a humanized version of the Hu177 antibody (D93). Serial blood samples were monitored for weekly time periods ranging from 6-20 weeks from 5 patients enrolled in this study. Serum were collected and stored in multiple aliquots at –20oC. Results: All 5 patients demonstrated an eventual lowering of shed epitopes, with the time course for a sustained reduction varying with each patient. Once a nadir in the Hu177 epitope occurred it would last from 4-6 weeks. In one patient (KL) the nadir levels were the most sustained and developed late in the course of treatment who had remarkable decreased changes in CA125 that was previously rapidly increasing. This patient had platinum resistant peritoneal carcinomatosis without any dominant masses. Upon failure of topotecan erlotinib as demonstrated by rising markers and development of ascites and a pleural effusion, the shed epitopes rapidly recurred starting at week 18 of the treatment. Comparable serial data were found with the humanized D93 antibody although the sensitivity was more pronounced with the Hu177 antibody. Conclusions: Our interpretation is that this Hu177 determination may provide an indication of active invasion of extracellular matrix (such as in the peritoneum) and may be a useful indicator of a biological effect. We cannot exclude at this point that erlotinib may be contributing to such effects by changes in the matrix. Supported by a grant from the Chemotherapy Foundation Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4617. doi:1538-7445.AM2012-4617