Ileana Cuevas

Meningioma Transcript Profiles Reveal Deregulated Notch Signaling Pathway

Meningiomas constitute the second most common central nervous system tumor, and yet relatively little is known about the molecular events that are important for the pathogenesis and malignant progression of these tumors. We have used serial analysis of gene expression to compare the transcriptomes of nonneoplastic meninges and meningiomas of all malignancy grades. A novel finding from this screen is the induction of three components of the Notch signaling pathway: the transcription factor, hairy and enhancer of Split1 (HES1) and two members of the Groucho/transducin-like enhancer of Split family of corepressors, TLE2 and TLE3. TLE corepressors interact and modulate the activity of a wide range of transcriptional regulatory systems, one of which is HES1. We have shown that the transcript and protein levels of HES1, the Notch2 and Notch1 receptors and the Jagged1 ligand are induced in meningiomas of all grades, whereas induction of TLE2 and TLE3 occurs specifically in higher-grade meningiomas. Meningioma cell lines express components of the Notch signaling pathway and an inhibitor of this pathway suppresses meningioma cell survival. These results suggest that deregulated expression of the Notch pathway is a critical event in meningioma pathogenesis and that modulation of this and potentially other signaling pathways by TLE corepressors leads to a more malignant phenotype.

Stromal regulation of vessel stability by MMP14 and TGFβ

Innate regulatory networks within organs maintain tissue homeostasis and facilitate rapid responses to damage. We identified a novel pathway regulating vessel stability in tissues that involves matrix metalloproteinase 14 (MMP14) and transforming growth factor beta 1 (TGFβ1). Whereas plasma proteins rapidly extravasate out of vasculature in wild-type mice following acute damage, short-term treatment of mice in vivo with a broad-spectrum metalloproteinase inhibitor, neutralizing antibodies to TGFβ1, or an activin-like kinase 5 (ALK5) inhibitor significantly enhanced vessel leakage. By contrast, in a mouse model of age-related dermal fibrosis, where MMP14 activity and TGFβ bioavailability are chronically elevated, or in mice that ectopically express TGFβ in the epidermis, cutaneous vessels are resistant to acute leakage. Characteristic responses to tissue damage are reinstated if the fibrotic mice are pretreated with metalloproteinase inhibitors or TGFβ signaling antagonists. Neoplastic tissues, however, are in a constant state of tissue damage and exhibit altered hemodynamics owing to hyperleaky angiogenic vasculature. In two distinct transgenic mouse tumor models, inhibition of ALK5 further enhanced vascular leakage into the interstitium and facilitated increased delivery of high molecular weight compounds into premalignant tissue and tumors. Taken together, these data define a central pathway involving MMP14 and TGFβ that mediates vessel stability and vascular response to tissue injury. Antagonists of this pathway could be therapeutically exploited to improve the delivery of therapeutics or molecular contrast agents into tissues where chronic damage or neoplastic disease limits their efficient delivery.

Endothelial cell migration and vascular endothelial growth factor expression are the result of loss of breast tissue polarity

Recruiting a new blood supply is a rate-limiting step in tumor progression. In a three-dimensional model of breast carcinogenesis, disorganized, proliferative transformed breast epithelial cells express significantly higher expression of angiogenic genes compared with their polarized, growth-arrested nonmalignant counterparts. Elevated vascular endothelial growth factor (VEGF) secretion by malignant cells enhanced recruitment of endothelial cells (EC) in heterotypic cocultures. Significantly, phenotypic reversion of malignant cells via reexpression of HoxD10, which is lost in malignant progression, significantly attenuated VEGF expression in a hypoxia-inducible factor 1alpha-independent fashion and reduced EC migration. This was due primarily to restoring polarity: forced proliferation of polarized, nonmalignant cells did not induce VEGF expression and EC recruitment, whereas disrupting the architecture of growth-arrested, reverted cells did. These data show that disrupting cytostructure activates the angiogenic switch even in the absence of proliferation and/or hypoxia and restoring organization of malignant clusters reduces VEGF expression and EC activation to levels found in quiescent nonmalignant epithelium. These data confirm the importance of tissue architecture and polarity in malignant progression.

HoxA5 Stabilizes Adherens Junctions Via Increased Akt1

Normal vascular development and angiogenesis is regulated by coordinated changes in cell-cell and cell-extracellular matrix (ECM) interactions. The Homeobox (Hox) family of transcription factors coordinately regulate expression of matrix degrading proteinases, integrins and ECM components and profoundly impact vascular remodeling. Whereas HoxA5 is downregulated in active angiogenic endothelial cells (EC), sustained expression of HoxA5 induces TSP-2 and blocks angiogenesis. Since HoxA5 is also lacking in EC in proliferating hemangiomas, we investigated whether restoring expression of HoxA5 could normalize hemangioma cell morphology and/or behavior. Sustained expression of HoxA5 in the murine hemangioma cell line (EOMA) reduced their growth in vivo and promoted branching morphogenesis in 3D BM cultures. Moreover, restoring HoxA5 expression increased the retention of β-catenin in adherens junctions and reduced permeability. In addition we also show that the HoxA5 mediated increase in stability of adherens junctions requires Akt1 activity and introduction of constitutively active myr-Akt in EOMA cells also increased retention of ß-catenin in adherens junctions. Finally we show that HoxA5 increases Akt1 mRNA, protein expression and further enhances Akt activity via a coordinate down regulation of PTEN. Together these results demonstrate a central role for HoxA5 in coordinating a stable vascular phenotype.

Restoring Transcription Factor HoxA5 Expression Inhibits the Growth of Experimental Hemangiomas in the Brain

Hemangiomas are angiogenesis-dependent benign vascular tumors that can rupture and cause intracranial hemorrhages. We previously showed that the transcription factor homeobox A5 (HoxA5), which is absent in activated angiogenic endothelial cells can block angiogenesis. Here, we investigated whether restoring expression of HoxA5 blocks hemangioma growth by transplanting mouse hemangioendothelioma endothelial cells (EOMA) or HoxA5-expressing EOMA cells into the brains of mice. The EOMA cells induced brain hemangiomas characterized by large cystlike spaces lined by thin walls of endothelial cells surrounded by scant smooth muscle cells. When HoxA5-expressing EOMA cells were injected, lesion volumes were reduced between 5- and 20-fold compared with the EOMA control group (p < 0.05). Restoration of HoxA5 was associated with increased thrombospondin-2, which inhibits angiogenesis and reduced hypoxia-inducible factor 1&agr; expression. These data suggest that restoring HoxA5 can attenuate experimental brain hemangioma development.

Chapter 2 Managing Tumor Angiogenesis: Lessons from VEGF-Resistant Tumors and Wounds

It is now well established both experimentally and clinically, that new blood vessel growth is required for tumors to grow beyond a few millimeters and metastasize [Folkman, J. (1995). In: Mendelsohn, L., Howley, P., Israel, A. (Eds.), The Molecular Basis of Cancer, WB Saunders Company, Philadelphia, pp. 206-225]. Angiogenesis, the process of forming new blood vessels from preexisting vessels, provides the tumor with additional oxygen and nutrients for its continued growth. In addition, the proximity and increase in vascular density enhance the likelihood of tumor cells entering the bloodstream to eventually metastasize. Since the initial observations of Dr. Folkman in the late 1970s, research over the past 30 years has focused intensely on identifying points in which the angiogenic cycle can be disrupted and has become an important component of current therapies to limit tumor progression.

Sustained Endothelial Expression of HoxA5 In Vivo Impairs Pathological Angiogenesis And Tumor Progression

HoxA5 is expressed in quiescent endothelial cells (EC), but absent in activated angiogenic EC. To examine the efficacy of targeting HoxA5 therapeutically to quell pathologic or tumor angiogenesis, we generated an inducible, transgenic mouse model of sustained HoxA5 expression in ECs. During pathologic angiogenesis, sustained HoxA5 regulates expression several angiogenic effector molecules, notably increased expression of TSP-2 and reduced expression of VEGF, thus leading to inhibition of pathological angiogenesis in tissues. To evaluate if this impressive reduction of vascularization could also impact tumor angiogenesis, HoxA5 mice were bred with a mouse model of de novo squamous carcinogenesis, e.g., K14-HPV16 mice. Activation of EC-HoxA5 significantly reduced infiltration by mast cells into neoplastic skin, an early hallmark of progression to dysplasia, reduced angiogenic vasculature, and blunted characteristics of tumor progression. To evaluate HoxA5 as a therapeutic, topical application of a HoxA5 transgene onto early neoplastic skin of K14-HPV16 mice similarly resulted in a significant impairment of angiogenic vasculature and progression to dysplasia to a similar extent as observed with genetic delivery of HoxA5. Together these data indicate that HoxA5 represents a novel molecule for restricting pathological and tumorigenic angiogenesis.

Abstract A07: Breast tumor microenvironment shapes vascular response to endothelial HoxA5 expression

Current anti-angiogenic therapy for the treatment of solid tumors is based on directed inhibition of growth factor signaling pathways essential for the development of new blood vessels. Despite evidence showing a positive correlation between angiogenesis and breast cancer progression, existing anti-angiogenic therapies have not proven effective at clinically managing breast tumors or prolonging patient survival. Several studies have shown that tissue microenvironment shapes local angiogenic responses and tumor progression - a finding that may partially explain the refractoriness of breast tumors to anti-angiogenic therapy. Data from our laboratory supports an anti-angiogenic role for the HoxA5 homeodomain containing transcription factor. Using the KRT14-HPV16 mouse model of skin cancer crossed with our tetracycline-regulated mouse line that expresses HoxA5 in the endothelium, we observed that HoxA5 reduced angiogenesis and slowed tumor progression in the skin. Thus, we hypothesized that constitutive endothelial expression of HoxA5 during mammary tumor development would prevent tumor growth and metastasis via modulation of the endothelial phenotype. Surprisingly, in the MMTV-PyMT mouse model of breast cancer, we observed that endothelial HoxA5 expression increased both primary tumor growth and lung metastasis. Mammary tumors from PyMT/HoxA5+ mice had larger areas of hypoxia and necrosis, as compared to primary tumors from control mice. Although we did not detect any change in intra-tumoral vascular staining (CD31+), we observed an increased number of large vessels and reduced vascular leakage in the primary tumors from PyMT/HoxA5+ mice. In contrast, subcutaneous injection of MMTV-PyMT mammary tumor cells into HoxA5 transgenic mice displayed significantly reduced tumor growth and decreased intra-tumoral vascularization, as compared to controls. This suggests that, unlike the anti-angiogenic properties of HoxA5 in the skin microenvironment, within the context of the mammary gland HoxA5 has vascular normalization effects. We conclude that the tissue microenvironment shapes the vascular response to anti-angiogenic agents and thereby controls tumor progression. Citation Format: Josette Northcott, Ileana Cuevas, Hans Layman, Nancy Boudreau. Breast tumor microenvironment shapes vascular response to endothelial HoxA5 expression. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A07. doi:10.1158/1538-7445.CHTME14-A07