Ets1 and Ets2 are required for endothelial cell survival during embryonic angiogenesis.

The ras/Raf/Mek/Erk pathway plays a central role in coordinating endothelial cell activities during angiogenesis. Transcription factors Ets1 and Ets2 are targets of ras/Erk signaling pathways that have been implicated in endothelial cell function in vitro, but their precise role in vascular formation and function in vivo remains ill-defined. In this work, mutation of both Ets1 and Ets2 resulted in embryonic lethality at midgestation, with striking defects in vascular branching having been observed. The action of these factors was endothelial cell autonomous as demonstrated using Cre/loxP technology. Analysis of Ets1/Ets2 target genes in isolated embryonic endothelial cells demonstrated down-regulation of Mmp9, Bcl-X(L), and cIAP2 in double mutants versus controls, and chromatin immunoprecipitation revealed that both Ets1 and Ets2 were loaded at target promoters. Consistent with these observations, endothelial cell apoptosis was significantly increased both in vivo and in vitro when both Ets1 and Ets2 were mutated. These results establish essential and overlapping functions for Ets1 and Ets2 in coordinating endothelial cell functions with survival during embryonic angiogenesis.

Study of the biologic effects of lapatinib, a reversible inhibitor of ErbB1 and ErbB2 tyrosine kinases, on tumor growth and survival pathways in patients with advanced malignancies.

PURPOSE: This was a pilot study to assess the biologic effects of lapatinib on various tumor growth/survival pathways in patients with advanced ErbB1 and/or ErbB2-overexpressing solid malignancies. PATIENTS AND METHODS: Heavily pretreated patients with metastatic cancers overexpressing ErbB2 and/or expressing ErbB1 were randomly assigned to one of five dose cohorts of lapatinib (GW572016) administered orally once daily continuously. The biologic effects of lapatinib on tumor growth and survival pathways were assessed in tumor biopsies obtained before and after 21 days of therapy. Clinical response was determined at 8 weeks. RESULTS: Sequential tumor biopsies from 33 patients were examined. Partial responses occurred in four patients with breast cancer, and disease stabilization occurred in 11 others with various malignancies. Responders exhibited variable levels of inhibition of p-ErbB1, p-ErbB2, p-Erk1/2, p-Akt, cyclin D1, and transforming growth factor alpha. Even some nonresponders demonstrated varying degrees of biomarker inhibition. Increased tumor cell apoptosis (TUNEL) occurred in patients with evidence of tumor regression but not in nonresponders (progressive disease). Clinical response was associated with a pretreatment TUNEL score > 0 and increased pretreatment expression of ErbB2, p-ErbB2, Erk1/2, p-Erk1/2, insulin-like growth factor receptor-1, p70 S6 kinase, and transforming growth factor alpha compared with nonresponders. CONCLUSION: Lapatinib exhibited preliminary evidence of biologic and clinical activity in ErbB1 and/or ErbB2-overexpressing tumors. However, the limited sample size of this study and the variability of the biologic endpoints suggest that further work is needed to prioritize biomarkers for disease-directed studies, and underscores the need for improved trial design strategies in early clinical studies of targeted agents.

Activated Ets2 is required for persistent inflammatory responses in the motheaten viable model.

The Ets2 transcription factor is constitutively phosphorylated on residue Thr(72) in macrophages derived from mice homozygous for the motheaten viable (me-v) allele of the hemopoietic cell phosphatase (Hcph) gene. To genetically test the importance of signaling through residue Thr(72) of Ets2 during inflammation, the Ets2(A72) mutant allele, which cannot be phosphorylated on Thr(72), was combined with the Hcph(me-v) allele in mice. Ets2(A72/A72) moderated the inflammation-related pathology of Hcph(me-v/me-v) mice, as demonstrated by the increased life span and the decreased macrophage infiltration in skin and lungs of these mice. Macrophage apoptosis induced by cytokine withdrawal was also increased in the double-mutant mice. Importantly, the Ets2(A72/A72) allele resulted in decreased expression of inflammatory response genes, including TNF-alpha, CCL3, matrix metalloprotease 9, integrin alpha(M), and Bcl-X in alveolar macrophage. Ets2 phosphorylation was required for persistent activation of TNF-alpha following LPS stimulation of bone marrow-derived macrophages. The phosphorylation of Ets2 functions in the severe inflammatory phenotype of the me-v model by mediating both macrophage survival and inflammatory gene expression.

Ets2-dependent stromal regulation of mouse mammary tumors.

The Ets2 transcription factor is regulated by mitogen-activated protein (MAP) kinase phosphorylation of a single threonine residue. We generated by gene targeting a single codon mutation in Ets2 substituting Ala for the critical Thr-72 phosphorylation site (Ets2A72), to investigate the importance of MAP kinase activation of Ets2 in embryo and tumor development. Ets2(A72/A72) mice are viable and develop normally. However, combining the Ets2A72 allele with a deletion mutant of Ets2 results in lethality at E11.5 and shows that Ets2A72 is a hypomorphic allele. Mammary tumors caused by transgenic polyomavirus middle T antigen, activated Neu(Erbb2), or the combination of Neu and transgenic VEGF (Neu; VEGF-25) were all restricted in Ets2(A72/A72) females. The Ets2(A72/A72) restriction on Neu; VEGF-25 tumor growth was associated with increased p21Cip1 expression. The size of tumors transplanted into fat pads of mice with Ets2 targeted alleles was correlated directly with Ets2 activity and fewer stromal cells expressing matrix metalloproteinase 9 (MMP-9). Decreased MMP-3 and MMP-9 mRNAs were confirmed in Ets2(A72/A72) macrophages. Activation of Ets2 at Thr-72 acts in the stroma, downstream of vascular endothelial growth factor production, in part through the regulation of macrophage proteases to support the progression of Neu- and polyomavirus middle-T-initiated mammary tumors.