Temporal multi-omics identifies LRG1 as a vascular niche instructor of metastasis.

Metastasis is the primary cause of cancer-related mortality. Tumor cell interactions with cells of the vessel wall are decisive and potentially rate-limiting for metastasis. The molecular nature of this cross-talk is, beyond candidate gene approaches, hitherto poorly understood. Using endothelial cell (EC) bulk and single-cell transcriptomics in combination with serum proteomics, we traced the evolution of the metastatic vascular niche in surgical models of lung metastasis. Temporal multiomics revealed that primary tumors systemically reprogram the body's vascular endothelium to perturb homeostasis and to precondition the vascular niche for metastatic growth. The vasculature with its enormous surface thereby serves as amplifier of tumor-induced instructive signals. Comparative analysis of lung EC gene expression and secretome identified the transforming growth factor­ß (TGFß) pathway specifier LRG1, leucine-rich alpha-2-glycoprotein 1, as an early instructor of metastasis. In the presence of a primary tumor, ECs systemically up-regulated LRG1 in a signal transducer and activator of transcription 3 (STAT3)­dependent manner. A meta-analysis of retrospective clinical studies revealed a corresponding up-regulation of LRG1 concentrations in the serum of patients with cancer. Functionally, systemic up-regulation of LRG1 promoted metastasis in mice by increasing the number of prometastatic neural/glial antigen 2 (NG2)+ perivascular cells. In turn, genetic deletion of Lrg1 hampered growth of lung metastasis. Postsurgical adjuvant administration of an LRG1-neutralizing antibody delayed metastatic growth and increased overall survival. This study has established a systems map of early primary tumor-induced vascular changes and identified LRG1 as a therapeutic target for metastasis.

ASK1 suppresses NK cell-mediated intravascular tumor cell clearance in lung metastasis.

Tumor metastasis is the leading cause of death worldwide and involves an extremely complex process composed of multiple steps. Our previous study demonstrated that apoptosis signal-regulating kinase 1 (ASK1) deficiency in mice attenuates tumor metastasis in an experimental lung metastasis model. However, the steps of tumor metastasis regulated by ASK1 remain unclear. Here, we showed that ASK1 deficiency in mice promotes natural killer (NK) cell-mediated intravascular tumor cell clearance in the initial hours of metastasis. In response to tumor inoculation, ASK1 deficiency upregulated immune response-related genes, including interferon-gamma (IFNγ). We also revealed that NK cells are required for these anti-metastatic phenotypes. ASK1 deficiency augmented cytokine production chemoattractive to NK cells possibly through induction of the ligand for NKG2D, a key activating receptor of NK cells, leading to further recruitment of NK cells into the lung. These results indicate that ASK1 negatively regulates NK cell-dependent anti-tumor immunity and that ASK1-targeted therapy can provide a new tool for cancer immunotherapy to overcome tumor metastasis.

Alternatively Spliced Form of Angiopoietin-2 as a New Vascular Rheostat.

Angiopoietin (ANPGT)-TIE signaling serves as a critical regulator of vessel maturation controlling vascular quiescence, maintenance, and homeostasis (primarily through ANGPT1-TIE2 signaling), as well as enabling vascular plasticity and responsiveness to exogenous cytokines (primarily through antagonistically acting ANGPT2). An alternatively spliced form of ANGPT2 (ANGPT2443) was first reported 20 years ago. Yet, little is known to this day about its biological functions. In this issue of Cancer Research, Kapiainen and colleagues report an elegant series of experiments adding to the complexity and contextuality of ANGPT-TIE signaling. The authors studied the function of ANGPT2443 in cellular experiments as well as in a genetic model in vivo, revealing that it is proteolytically cleaved into a lower molecular weight isoform (termed ANGPT2DAP) that lacks the superclustering domain necessary for multimer formation. When compared with full-length ANGPT2, ANGPT2443 and ANGPT2DAP showed lower binding affinity to α5ß1 integrin, but were more potent inhibitors of ANGPT1-TIE2 signaling. Functionally, ANGPT2443 impaired vessel enlargement and vein morphogenesis during postnatal retinal angiogenesis. Tumor experiments in Angpt2443-expressing mice showed enhanced destabilization of the lung vasculature, with varying effects on metastasis. Taken together, the study provides important insight into the significance of ANGPT2 alternative splicing and identifies ANGPT2443 and ANGPT2DAP as a biological rheostat of ANGPT1-TIE2 signaling. Future work will need to characterize the relative ratios and functional contributions of the ANGPT2 variants in different pathophysiologic settings.See related article by Kapiainen et al., p. 129.

Preclinical validation of a novel metastasis-inhibiting Tie1 function-blocking antibody.

The angiopoietin (Ang)-Tie pathway has been intensely pursued as candidate second-generation anti-angiogenic target. While much of the translational work has focused on the ligand Ang2, the clinical efficacy of Ang2-targeting drugs is limited and failed to improve patient survival. In turn, the orphan receptor Tie1 remains therapeutically unexplored, although its endothelial-specific genetic deletion has previously been shown to result in a strong reduction in metastatic growth. Here, we report a novel Tie1 function-blocking antibody (AB-Tie1-39), which suppressed postnatal retinal angiogenesis. During primary tumor growth, neoadjuvant administration of AB-Tie1-39 strongly impeded systemic metastasis. Furthermore, the administration of AB-Tie1-39 in a perioperative therapeutic window led to a significant survival advantage as compared to control-IgG-treated mice. Additional in vivo experimental metastasis and in vitro transmigration assays concurrently revealed that AB-Tie1-39 treatment suppressed tumor cell extravasation at secondary sites. Taken together, the data phenocopy previous genetic work in endothelial Tie1 KO mice and thereby validate AB-Tie1-39 as a Tie1 function-blocking antibody. The study establishes Tie1 as a therapeutic target for metastasis in a perioperative or neoadjuvant setting.

[Functional Analysis of Apoptosis Signal-regulating Kinase Family in a Murine Model of Tumor Metastasis].

Stress-responsive signaling pathways convert cellular stresses into various physiological responses, such as cell proliferation, apoptosis, and inflammation. Signal pathway dysfunction thus induces abnormal cellular behaviors that may lead to tumorigenesis and tumor progression, including metastasis. Tumor metastasis is the spread of tumor cells from primary lesions to other distant tissues/organs. Several types of murine model which mimic the progression of human cancer have been established for preclinical studies to understand the biology of cancer. Mitogen-activated protein kinase (MAPK) cascades are one of the stress-responsive signaling pathways and are intricately involved in both tumor promotion and suppression. Here, we present the diverse roles of apoptosis signal-regulating kinase (ASK) family molecules in tumor formation and progression. ASK family is a member of MAPK kinase kinase (MAP3K) family in the c-Jun N-terminal kinase (JNK) and p38 MAPK pathways and comprises three family members, ASK1, ASK2, and ASK3. Accumulating evidence indicates that ASK1 controls tumorigenesis through the regulation of innate immunity and apoptosis. ASK2 also regulates tumorigenesis via apoptosis. Furthermore, analysis of the experimental lung metastasis model in mice suggests that host ASK1 deficiency attenuates tumor lung metastasis. In this symposium review, we discuss the potential roles of ASK family in the context of tumor metastasis.

ASK1 facilitates tumor metastasis through phosphorylation of an ADP receptor P2Y12 in platelets.

Tumor metastasis is the major cause of deaths in cancer patients and is modulated by intertwined stress-responsive signaling cascades. Here we demonstrate that deletion of stress-responsive apoptosis signal-regulating kinase 1 (Ask1) in platelets results in unstable hemostasis and drastic attenuation of tumor lung metastasis, both of which are attributable to platelet dysfunction. Platelet-specific deletion of Ask1 in mice leads to defects in ADP-dependent platelet aggregation, unstable hemostasis and subsequent attenuation of tumor metastasis. We also revealed that activating phosphorylation of Akt is attenuated in Ask1-deficient platelets, contrary to the previous reports suggesting that Akt is negatively regulated by ASK1. Mechanistically, ASK1-JNK/p38 axis phosphorylates an ADP receptor P2Y12 at Thr345, which is required for the ADP-dependent sustained Akt activity that is vital to normal platelet functions. Our findings offer insight into positive regulation of Akt signaling through P2Y12 phosphorylation as well as MAPK signaling in platelets by ASK1 and suggest that ASK1-JNK/p38 axis provides a new therapeutic opportunity for tumor metastasis.

ASK family and cancer.

Cancer is a major problem in public health and is one of the leading causes of mortality worldwide. Many types of cancer cells exhibit aberrant cellular signal transduction in response to stress, which often leads to oncogenesis. Mitogen-activated protein kinase (MAPK) signal cascades are one of the important intracellular stress signaling pathways closely related to cancer. The key molecules in MAPK signal cascades that respond to various types of stressors are apoptosis signal-regulating kinase (ASK) family members; ASK1, ASK2 and ASK3. ASK family members are activated by a wide variety of stressors, and they regulate various cellular responses, such as cell proliferation, inflammation and apoptosis. In this review, we will discuss both the oncogenic and anti-oncogenic roles of the ASK family members in various contexts of cancer development with deeper insights into the involvement of ASK family members in cancer pathology.

In vivo gene manipulation reveals the impact of stress-responsive MAPK pathways on tumor progression.

It has been widely accepted that tumor cells and normal stromal cells in the host environment coordinately modulate tumor progression. Mitogen-activated protein kinase pathways are the representative stress-responsive cascades that exert proper cellular responses to divergent environmental stimuli. Genetically engineered mouse models and chemically induced tumorigenesis models have revealed that components of the MAPK pathway not only regulate the behavior of tumor cells themselves but also that of surrounding normal stromal cells in the host environment during cancer pathogenesis. The individual functions of MAPK pathway components in tumor initiation and progression vary depending on the stimuli and the stromal cell types involved in tumor progression, in addition to the molecular isoforms of the components and the origins of the tumor. Recent studies have indicated that MAPK pathway components synergize with environmental factors (e.g. tobacco smoke and diet) to affect tumor initiation and progression. Moreover, some components play distinct roles in the course of tumor progression, such as before and after the establishment of tumors. Hence, a comprehensive understanding of the multifaceted functions of MAPK pathway components in tumor initiation and progression is essential for the improvement of cancer therapy. In this review, we focus on the reports that utilized knockout, conditional knockout, and transgenic mice of MAPK pathway components to investigate the effects of MAPK pathway components on tumor initiation and progression in the host environment.

Rhomboid protease PARL mediates the mitochondrial membrane potential loss-induced cleavage of PGAM5.

Regulated intramembrane proteolysis is a widely conserved mechanism for controlling diverse biological processes. Considering that proteolysis is irreversible, it must be precisely regulated in a context-dependent manner. Here, we show that phosphoglycerate mutase 5 (PGAM5), a mitochondrial Ser/Thr protein phosphatase, is cleaved in its N-terminal transmembrane domain in response to mitochondrial membrane potential (ΔΨ(m)) loss. This ΔΨ(m) loss-dependent cleavage of PGAM5 was mediated by presenilin-associated rhomboid-like (PARL). PARL is a mitochondrial resident rhomboid serine protease and has recently been reported to mediate the cleavage of PINK1, a mitochondrial Ser/Thr protein kinase, in healthy mitochondria with intact ΔΨ(m). Intriguingly, we found that PARL dissociated from PINK1 and reciprocally associated with PGAM5 in response to ΔΨ(m) loss. These results suggest that PARL mediates differential cleavage of PINK1 and PGAM5 depending on the health status of mitochondria. Our data provide a prototypical example of stress-dependent regulation of PARL-mediated regulated intramembrane proteolysis.