Erratum: Specific targeting of PDGFRß in the stroma inhibits growth and angiogenesis in tumors with high PDGF-BB expression: Erratum.

[This corrects the article DOI: 10.7150/thno.37851.].

Specific targeting of PDGFRß in the stroma inhibits growth and angiogenesis in tumors with high PDGF-BB expression.

PDGF-BB/PDGFRß signaling plays an important role during vascularization by mediating pericyte recruitment to the vasculature, promoting the integrity and function of vessels. Until now it has not been possible to assess the specific role of PDGFRß signaling in tumor progression and angiogenesis due to lack of appropriate animal models and molecular tools. Methods: In the present study, we used a transgenic knock-in mouse strain carrying a silent mutation in the PDGFRß ATP binding site that allows specific targeting of PDGFRß using the compound 1-NaPP1. To evaluate the impact of selective PDGFRß inhibition of stromal cells on tumor growth we investigated four tumor cell lines with no or low PDGFRß expression, i.e. Lewis lung carcinoma (LLC), EO771 breast carcinoma, B16 melanoma and a version of B16 that had been engineered to overexpress PDGF-BB (B16/PDGF-BB). Results: We found that specific impairment of PDGFRß kinase activity by 1-NaPP1 treatment efficiently suppressed growth in tumors with high expression of PDGF-BB, i.e. LLC and B16/PDGF-BB, while the clinically used PDGFRß kinase inhibitor imatinib did not suppress tumor growth. Notably, tumors with low levels of PDGF-BB, i.e. EO771 and B16, neither responded to 1-NaPP1 nor to imatinib treatment. Inhibition of PDGFRß by either drug impaired tumor vascularization and also affected pericyte coverage; however, specific targeting of PDGFRß by 1-NaPP1 resulted in a more pronounced decrease in vessel function with increased vessel apoptosis in high PDGF-BB expressing tumors, compared to treatment with imatinib. In vitro analysis of PDGFRß ASKA mouse embryo fibroblasts and the mesenchymal progenitor cell line 10T1/2 revealed that PDGF-BB induced NG2 expression, consistent with the in vivo data. Conclusion: Specific targeting of PDGFRß signaling significantly inhibits tumor progression and angiogenesis depending on PDGF-BB expression. Our data suggest that targeting PDGFRß in the tumor stroma could have therapeutic value in patients with high tumor PDGF-BB expression.

Exposure to EGF and 17ß­estradiol irreversibly affects the proliferation and transformation of MCF7 cells but is not sufficient to promote tumor growth in a xenograft mouse model upon withdrawal of exposure.

Epidermal growth factor (EGF) and estrogen are potent regulators of breast tumorigenesis. Their short­term actions on human breast epithelial cells have been investigated extensively. However, the consequence of a long­term exposure to EGF and estrogen remains to be fully elucidated. The present study examined the effects of long­term exposure to EGF and 17ß­estradiol on the proliferation, transformation, expression of markers of stemness, and tumorigenesis of MCF7 human breast adenocarcinoma cells. Exposure to EGF and/or 17ß­estradiol irreversibly enhanced the proliferation rate of MCF7 cells, even following withdrawal. However, in a mouse xenograft experiment, no significant difference in tumor volume was observed between tumors derived from cells exposed to EGF, 17ß­estradiol or EGF + 17ß­estradiol. Immunohistochemistry performed on tumors derived from 17ß­estradiol­exposed cells revealed reduced cell proliferation and vessel scores, according to the results obtained using Ki67 and von Willebrand factor staining, respectively. The EGF­ and/or 17ß­estradiol­treated cells exhibited an increased ratio of cluster of differentiation (CD)44+/CD24­ cells and enhanced ability to form mammospheres. Furthermore, the long­term exposure of MCF7 cells to EGF and 17ß­estradiol altered their responsiveness to short­term stimulatory or inhibitory treatments with EGF, 17ß­estradiol, transforming growth factor­ß1 (TGFß1), Iressa and SB431542. Therefore, the findings indicated that sustained exposure of MCF7 cells to EGF and/or 17ß­estradiol resulted in enhanced cell proliferation and mammosphere formation, an increased ratio of CD44+/CD24­ cells, and altered responses to short­term treatments with EGF, 17ß­estradiol, TGFß1, and drugs inhibiting these signaling pathways. However, this sustained exposure was not sufficient to affect tumor take or volume in a xenograft mouse model.

VE-Cadherin-Mediated Epigenetic Regulation of Endothelial Gene Expression.

RATIONALE: The mechanistic foundation of vascular maturation is still largely unknown. Several human pathologies are characterized by deregulated angiogenesis and unstable blood vessels. Solid tumors, for instance, get their nourishment from newly formed structurally abnormal vessels which present wide and irregular interendothelial junctions. Expression and clustering of the main endothelial-specific adherens junction protein, VEC (vascular endothelial cadherin), upregulate genes with key roles in endothelial differentiation and stability. OBJECTIVE: We aim at understanding the molecular mechanisms through which VEC triggers the expression of a set of genes involved in endothelial differentiation and vascular stabilization. METHODS AND RESULTS: We compared a VEC-null cell line with the same line reconstituted with VEC wild-type cDNA. VEC expression and clustering upregulated endothelial-specific genes with key roles in vascular stabilization including claudin-5, vascular endothelial-protein tyrosine phosphatase (VE-PTP), and von Willebrand factor (vWf). Mechanistically, VEC exerts this effect by inhibiting polycomb protein activity on the specific gene promoters. This is achieved by preventing nuclear translocation of FoxO1 (Forkhead box protein O1) and ß-catenin, which contribute to PRC2 (polycomb repressive complex-2) binding to promoter regions of claudin-5, VE-PTP, and vWf. VEC/ß-catenin complex also sequesters a core subunit of PRC2 (Ezh2 [enhancer of zeste homolog 2]) at the cell membrane, preventing its nuclear translocation. Inhibition of Ezh2/VEC association increases Ezh2 recruitment to claudin-5, VE-PTP, and vWf promoters, causing gene downregulation. RNA sequencing comparison of VEC-null and VEC-positive cells suggested a more general role of VEC in activating endothelial genes and triggering a vascular stability-related gene expression program. In pathological angiogenesis of human ovarian carcinomas, reduced VEC expression paralleled decreased levels of claudin-5 and VE-PTP. CONCLUSIONS: These data extend the knowledge of polycomb-mediated regulation of gene expression to endothelial cell differentiation and vessel maturation. The identified mechanism opens novel therapeutic opportunities to modulate endothelial gene expression and induce vascular normalization through pharmacological inhibition of the polycomb-mediated repression system.

Deregulated TGF-ß/BMP Signaling in Vascular Malformations.

Correct organization of the vascular tree requires the balanced activities of several signaling pathways that regulate tubulogenesis and vascular branching, elongation, and pruning. When this balance is lost, the vessels can be malformed and fragile, and they can lose arteriovenous differentiation. In this review, we concentrate on the transforming growth factor (TGF)-ß/bone morphogenetic protein (BMP) pathway, which is one of the most important and complex signaling systems in vascular development. Inactivation of these pathways can lead to altered vascular organization in the embryo. In addition, many vascular malformations are related to deregulation of TGF-ß/BMP signaling. Here, we focus on two of the most studied vascular malformations that are induced by deregulation of TGF-ß/BMP signaling: hereditary hemorrhagic telangiectasia (HHT) and cerebral cavernous malformation (CCM). The first of these is related to loss-of-function mutation of the TGF-ß/BMP receptor complex and the second to increased signaling sensitivity to TGF-ß/BMP. In this review, we discuss the potential therapeutic targets against these vascular malformations identified so far, as well as their basis in general mechanisms of vascular development and stability.

Compound genetically engineered mouse models of cancer reveal dual targeting of ALK1 and endoglin as a synergistic opportunity to impinge on angiogenic TGF-ß signaling.

Angiogenesis occurs early in tumor development, sustains primary tumor growth and provides a route for metastatic escape. The TGF-ß family receptors modulate angiogenesis via endothelial-cell specific pathways. Here we investigate the interaction of two such receptors, ALK1 and endoglin, in pancreatic neuroendocrine tumors (PanNET). Independently, ALK1 and endoglin deficiencies exhibited genetically divergent phenotypes, while both highly correlate to an endothelial metagene in human and mouse PanNETs. A concurrent deficiency of both receptors synergistically decreased tumor burden to a greater extent than either individual knockdown. Furthermore, the knockout of Gdf2 (BMP9), the primary ligand for ALK1 and endoglin, exhibited a mixed phenotype from each of ALK1 and endoglin deficiencies; overall primary tumor burden decreased, but hepatic metastases increased. Tumors lacking BMP9 display a hyperbranching vasculature, and an increase in vascular mesenchymal-marker expression, which may be implicit in the increase in metastases. Taken together, our work cautions against singular blockade of BMP9 and instead demonstrates the utility of dual blockade of ALK1 and endoglin as a strategy for anti-angiogenic therapy in PanNET.

Functional Characterization of Germline Mutations in PDGFB and PDGFRB in Primary Familial Brain Calcification.

Primary Familial Brain Calcification (PFBC), a neurodegenerative disease characterized by progressive pericapillary calcifications, has recently been linked to heterozygous mutations in PDGFB and PDGFRB genes. Here, we functionally analyzed several of these mutations in vitro. All six analyzed PDGFB mutations led to complete loss of PDGF-B function either through abolished protein synthesis or through defective binding and/or stimulation of PDGF-Rß. The three analyzed PDGFRB mutations had more diverse consequences. Whereas PDGF-Rß autophosphorylation was almost totally abolished in the PDGFRB L658P mutation, the two sporadic PDGFRB mutations R987W and E1071V caused reductions in protein levels and specific changes in the intensity and kinetics of PLCγ activation, respectively. Since at least some of the PDGFB mutations were predicted to act through haploinsufficiency, we explored the consequences of reduced Pdgfb or Pdgfrb transcript and protein levels in mice. Heterozygous Pdgfb or Pdgfrb knockouts, as well as double Pdgfb+/-;Pdgfrb+/- mice did not develop brain calcification, nor did Pdgfrbredeye/redeye mice, which show a 90% reduction of PDGFRß protein levels. In contrast, Pdgfbret/ret mice, which have altered tissue distribution of PDGF-B protein due to loss of a proteoglycan binding motif, developed brain calcifications. We also determined pericyte coverage in calcification-prone and non-calcification-prone brain regions in Pdgfbret/ret mice. Surprisingly and contrary to our hypothesis, we found that the calcification-prone brain regions in Pdgfbret/ret mice model had a higher pericyte coverage and a more intact blood-brain barrier (BBB) compared to non-calcification-prone brain regions. While our findings provide clear evidence that loss-of-function mutations in PDGFB or PDGFRB cause PFBC, they also demonstrate species differences in the threshold levels of PDGF-B/PDGF-Rß signaling that protect against small-vessel calcification in the brain. They further implicate region-specific susceptibility factor(s) in PFBC pathogenesis that are distinct from pericyte and BBB deficiency.

Endothelial ALK1 Is a Therapeutic Target to Block Metastatic Dissemination of Breast Cancer.

Exploration of new strategies for the prevention of breast cancer metastasis is justifiably at the center of clinical attention. In this study, we combined a computational biology approach with mechanism-based preclinical trials to identify inhibitors of activin-like receptor kinase (ALK) 1 as effective agents for blocking angiogenesis and metastasis in breast cancer. Pharmacologic targeting of ALK1 provided long-term therapeutic benefit in mouse models of mammary carcinoma, accompanied by strikingly reduced metastatic colonization as a monotherapy or part of combinations with chemotherapy. Gene-expression analysis of breast cancer specimens from a population-based nested case-control study encompassing 768 subjects defined endothelial expression of ALK1 as an independent and highly specific prognostic factor for metastatic manifestation, a finding that was corroborated in an independent clinical cohort. Overall, our results suggest that pharmacologic inhibition of endothelial ALK1 constitutes a tractable strategy for interfering with metastatic dissemination of breast cancer.

Bone morphogenetic protein-9 inhibits lymphatic vessel formation via activin receptor-like kinase 1 during development and cancer progression.

Lymphatic vessels (LVs) play critical roles in the maintenance of fluid homeostasis and in pathological conditions, including cancer metastasis. Although mutations in ALK1, a member of the transforming growth factor (TGF)-ß/bone morphogenetic protein (BMP) receptor family, have been linked to hereditary hemorrhagic telangiectasia, a human vascular disease, the roles of activin receptor-like kinase 1 (ALK-1) signals in LV formation largely remain to be elucidated. We show that ALK-1 signals inhibit LV formation, and LVs were enlarged in multiple organs in Alk1-depleted mice. These inhibitory effects of ALK-1 signaling were mediated by BMP-9, which decreased the number of cultured lymphatic endothelial cells. Bmp9-deficient mouse embryos consistently exhibited enlarged dermal LVs. BMP-9 also inhibited LV formation during inflammation and tumorigenesis. BMP-9 downregulated the expression of the transcription factor prospero-related homeobox 1, which is necessary to maintain lymphatic endothelial cell identity. Furthermore, silencing prospero-related homeobox 1 expression inhibited lymphatic endothelial cell proliferation. Our findings reveal a unique molecular basis for the physiological and pathological roles of BMP-9/ALK-1 signals in LV formation.

Deficiency for endoglin in tumor vasculature weakens the endothelial barrier to metastatic dissemination.

Therapy-induced resistance remains a significant hurdle to achieve long-lasting responses and cures in cancer patients. We investigated the long-term consequences of genetically impaired angiogenesis by engineering multiple tumor models deprived of endoglin, a co-receptor for TGF-ß in endothelial cells actively engaged in angiogenesis. Tumors from endoglin-deficient mice adapted to the weakened angiogenic response, and refractoriness to diminished endoglin signaling was accompanied by increased metastatic capability. Mechanistic studies in multiple mouse models of cancer revealed that deficiency for endoglin resulted in a tumor vasculature that displayed hallmarks of endothelial-to-mesenchymal transition, a process of previously unknown significance in cancer biology, but shown by us to be associated with a reduced capacity of the vasculature to avert tumor cell intra- and extravasation. Nevertheless, tumors deprived of endoglin exhibited a delayed onset of resistance to anti-VEGF (vascular endothelial growth factor) agents, illustrating the therapeutic utility of combinatorial targeting of multiple angiogenic pathways for the treatment of cancer.

ALK1 as an emerging target for antiangiogenic therapy of cancer.

Members of the TGF-ß family act on many, if not all, cell types within the body, producing diverse and complex cellular outcomes. Activation of the endothelial cell-restricted TGF-ß type I receptor ALK1 results from the binding of several different ligands of the TGF-ß family, including bone morphogenetic protein (BMP) 9, BMP10, and TGF-ß. Mounting genetic, pharmacologic, and histopathologic evidence supports a critical role for ALK1 signaling in regulation of both developmental and pathologic blood vessel formation. However, the precise function of TGF-ß family signaling in endothelial cells is difficult to predict and appears highly context dependent because of the multitude of ligands and receptors influencing the final outcome. Pharmacologic inhibitors of ALK1 have recently been developed and will allow for more accurate studies of ALK1 function in vivo, as well as for assessment of ALK1 as a target for suppression of angiogenesis during tumor development. Herein, we will summarize the current view of ALK1 regulation of endothelial cell phenotype in vitro and in vivo as well as provide an outlook for the ongoing clinical trials of ALK1 inhibitors in malignant disease.

Genetic and pharmacological targeting of activin receptor-like kinase 1 impairs tumor growth and angiogenesis.

Members of the transforming growth factor beta (TGF-beta) family have been genetically linked to vascular formation during embryogenesis. However, contradictory studies about the role of TGF-beta and other family members with reported vascular functions, such as bone morphogenetic protein (BMP) 9, in physiological and pathological angiogenesis make the need for mechanistic studies apparent. We demonstrate, by genetic and pharmacological means, that the TGF-beta and BMP9 receptor activin receptor-like kinase (ALK) 1 represents a new therapeutic target for tumor angiogenesis. Diminution of ALK1 gene dosage or systemic treatment with the ALK1-Fc fusion protein RAP-041 retarded tumor growth and progression by inhibition of angiogenesis in a transgenic mouse model of multistep tumorigenesis. Furthermore, RAP-041 significantly impaired the in vitro and in vivo angiogenic response toward vascular endothelial growth factor A and basic fibroblast growth factor. In seeking the mechanism for the observed effects, we uncovered an unexpected signaling synergy between TGF-beta and BMP9, through which the combined action of the two factors augmented the endothelial cell response to angiogenic stimuli. We delineate a decisive role for signaling by TGF-beta family members in tumor angiogenesis and offer mechanistic insight for the forthcoming clinical development of drugs blocking ALK1 in oncology.