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Visualization of basal cell surface of ANGPT2-GFP HUVECs during cell spreading.

Supplementary Figure S1: Amino acid sequence of ANGPT2, ANGPT2443 and ANGTPT2DAP. Supplementary Figure S2: Competitive inhibition of ANGPT4-induced TIE2 phosphorylation by ANGPT2 isoforms. Supplementary Figure S3. TIE2 phosphorylation by ANGPT2 isoforms at the retracting cell edges. Supplementary Figure S4. Generation of Angpt2443 allele and the effects of Angpt2443 expression on the retina. Supplementary Figure S5. MMTV-PyMT mammary gland tumor model. Supplementary Figure S6. ANGPT2 at the Weibel-Palade bodies and the subendothelial space. Supplementary Figure S7. B16F10 melanoma lung colonization model. Supplementary Figure S8. E0771 breast cancer cell transplantation model.

Angiopoietin-2 (ANGPT2) is a context-dependent TIE2 agonistic or antagonistic ligand that induces diverse responses in cancer. Blocking ANGPT2 provides a promising strategy for inhibiting tumor growth and metastasis, yet variable effects of targeting ANGPT2 have complicated drug development. ANGPT2443 is a naturally occurring, lower oligomeric protein isoform whose expression is increased in cancer. Here, we use a knock-in mouse line (mice expressing Angpt2443), a genetic model for breast cancer and metastasis (MMTV-PyMT), a syngeneic melanoma lung colonization model (B16F10), and orthotopic injection of E0771 breast cancer cells to show that alternative forms increase the diversity of Angpt2 function. In a mouse retina model of angiogenesis, expression of Angpt2443 caused impaired venous development, suggesting enhanced function as a competitive antagonist for Tie2. In mammary gland tumor models, Angpt2443 differentially affected primary tumor growth and vascularization; these varying effects were associated with Angpt2 protein localization in the endothelium or in the stromal extracellular matrix as well as the frequency of Tie2-positive tumor blood vessels. In the presence of metastatic cells, Angpt2443 promoted destabilization of pulmonary vasculature and lung metastasis. In vitro, ANGPT2443 was susceptible to proteolytical cleavage, resulting in a monomeric ligand (ANGPT2DAP) that inhibited ANGPT1- or ANGPT4-induced TIE2 activation but did not bind to alternative ANGPT2 receptor α5β1 integrin. Collectively, these data reveal novel roles for the ANGPT2 N-terminal domain in blood vessel remodeling, tumor growth, metastasis, integrin binding, and proteolytic regulation.Significance:This study identifies the role of the N-terminal oligomerization domain of angiopoietin-2 in vascular remodeling and lung metastasis and provides new insights into mechanisms underlying the versatile functions of angiopoietin-2 in cancer.See related commentary by Kamiyama and Augustin, p. 35

The EGFR adaptor protein, CIN85, has been shown to promote breast cancer malignancy and hypoxia-inducible factor (HIF) stability. However, the mechanisms underlying cancer promotion remain ill defined. Here we show that CIN85 is a novel binding partner of the main HIF-prolyl hydroxylase, PHD2, but not of PHD1 or PHD3. Mechanistically, the N-terminal SRC homology 3 domains of CIN85 interacted with the proline-arginine–rich region within the N-terminus of PHD2, thereby inhibiting PHD2 activity and HIF degradation. This activity is essential in vivo, as specific loss of the CIN85–PHD2 interaction in CRISPR/Cas9-edited cells affected growth and migration properties, as well as tumor growth in mice. Overall, we discovered a previously unrecognized tumor growth checkpoint that is regulated by CIN85-PHD2 and uncovered an essential survival function in tumor cells by linking growth factor adaptors with hypoxia signaling.Significance:This study provides unprecedented evidence for an oxygen-independent mechanism of PHD2 regulation that has important implications in cancer cell survival.

Plasmids and site directed mutagenesis, GST-pull down, Fluorescence microscopy, CRISPR/Cas9-mediated EGLN1 gene editing, Lentivirus-mediated expression of sgRNAs and Cas9, Genomic DNA extraction and genotyping, RNA extraction and qRT-PCR, Scanning electron microscopy, Monolayer colony formation assay, Live Cell Imaging Assays Surface plasmon resonance (SPR)

Supplementary Tables I-III

Visualization of basal cell surface of GFP-HUVECs

Legends for Sup movies 1 and 2

Supplementary Materials and Methods

Supplementary figure 1. CIN85 interacts with PHD2 via three N-terminal SH3 domains. Supplementary figure 2. The amino acids 81-98 within the N-terminal region of PHD2 have a major impact on the interaction with CIN85. Supplementary figure 3. CRISPR/Cas9-mediated generation of MDA-MB-231 cells lacking the amino acids critical for interaction with CIN85. Supplementary figure 4. Loss of PHD2-CIN85 interaction does not influence HIF-1α and HIF-2α mRNA levels. Supplementary figure 5. Lack of the CIN85-PHD2 interaction in E10 and E12 cells mediates HIF-α resistance upon depletion of CIN85. Supplementary figure 6. Microscopy images of S, E10, and E12 cells. Supplementary figure 7. Live cell proliferation analysis of S, E10, and E12 cells. Supplementary figure 8. CRISPR/Cas9 mediated EGLN1 (PHD2) editing in MDA-MB-231 cells with EGLN1-sgRNA-219 and with EGLN1-sgRNA-292 did not show off-target effects.

The activation of initiator protein tissue factor (TF) is likely to be a crucial step in the blood coagulation process, which leads to fibrin formation. The stimuli responsible for inducing TF activation are largely undefined. Here we show that the oxidoreductase protein disulfide isomerase (PDI) directly promotes TF-dependent fibrin production during thrombus formation in vivo. After endothelial denudation of mouse carotid arteries, PDI was released at the injury site from adherent platelets and disrupted vessel wall cells. Inhibition of PDI decreased TF-triggered fibrin formation in different in vivo murine models of thrombus formation, as determined by intravital fluorescence microscopy. PDI infusion increased — and, under conditions of decreased platelet adhesion, PDI inhibition reduced — fibrin generation at the injury site, indicating that PDI can directly initiate blood coagulation. In vitro, human platelet–secreted PDI contributed to the activation of cryptic TF on microvesicles (microparticles). Mass spectrometry analyses indicated that part of the extracellular cysteine 209 of TF was constitutively glutathionylated. Mixed disulfide formation contributed to maintaining TF in a state of low functionality. We propose that reduced PDI activates TF by isomerization of a mixed disulfide and a free thiol to an intramolecular disulfide. Our findings suggest that disulfide isomerases can act as injury response signals that trigger the activation of fibrin formation following vessel injury.

The biopharmaceutical market is growing faster than ever, with two production systems competing for market dominance: mammalian cells and microorganisms. In recent years, based on the rise of antibody-based therapies, new biotherapeutic approvals have favored mammalian hosts. However, not only has extensive research elevated our understanding of microbes to new levels, but emerging therapeutic molecules also facilitate their use; thus, is it time for microbes to fight back? In this review, we answer this timely question by cross-comparing four microbial production hosts and examining the innovations made to both their secretion and post-translational modification (PTM) capabilities. Furthermore, we discuss the impact of tools, such as omics and systems biology, as well as alternative production systems and emerging biotherapeutics.