Tetsuro Masuda

Tumor cell-derived angiopoietin-like protein ANGPTL2 is a critical driver of metastasis

Strategies to inhibit metastasis have been mainly unsuccessful in part due to insufficient mechanistic understanding. Here, we report evidence of critical role for the angiopoietin-like protein 2 (ANGPTL2) in metastatic progression. In mice, Angptl2 has been implicated in inflammatory carcinogenesis but it has not been studied in human tumors. In patients with lung cancer, elevated levels of ANGPTL2 expression in tumor cells within the primary tumor were associated with a reduction in the period of disease-free survival after surgical resection. Transcription factors NFATc, ATF2, and c-Jun upregulated in aggressive tumor cells promoted increased Angptl2 expression. Most notably, tumor cell-derived ANGPTL2 increased in vitro motility and invasion in an autocrine/paracrine manner, conferring an aggressive metastatic tumor phenotype. In xenograft mouse models, tumor cell-derived ANGPTL2 accelerated metastasis and shortened survival whereas attenuating ANGPTL2 expression in tumor cells-blunted metastasis and extended survival. Overall, our findings showed that tumor cell-derived ANGPTL2 drives metastasis and provided an initial proof of concept for blockade of its action as a strategy to antagonize the metastatic process.

The Secreted Protein ANGPTL2 Promotes Metastasis of Osteosarcoma Cells Through Integrin α5β1, p38 MAPK, and Matrix Metalloproteinases

Preventing signaling by ANGPTL2, which is stimulated by the tumor microenvironment, could inhibit metastasis. Microenvironment Drives Osteosarcoma Metastasis The selective pressures of the tumor microenvironment alter the behavior of cancer cells. Odagiri et al. found that the expression of ANGPTL2, encoding the secreted angiopoietin-like protein 2, increased in osteosarcoma cells grown in xenografts in mice or cultured in conditions that mimic the tumor microenvironment. Silencing ANGPTL2 or overexpressing a proteolytically cleaved form decreased matrix metalloproteinase-9 (MMP-9) activity, delayed the onset of metastasis from xenografts, and prolonged survival in mice. The abundance of ANGPTL2 correlated with that of MMP-9 in patient samples, and both inversely correlated with metastasis-free survival in patients. The findings highlight the influence of the tumor microenvironment and implicate ANGPTL2 as a target to hinder metastasis in osteosarcoma. The tumor microenvironment can enhance the invasive capacity of tumor cells. We showed that expression of angiopoietin-like protein 2 (ANGPTL2) in osteosarcoma (OS) cell lines increased and the methylation of its promoter decreased with time when grown as xenografts in mice compared with culture. Compared with cells grown in normal culture conditions, the expression of genes encoding DNA demethylation–related enzymes increased in tumor cells implanted into mice or grown in hypoxic, serum-starved culture conditions. ANGPTL2 expression in OS cell lines correlated with increased tumor metastasis and decreased animal survival by promoting tumor cell intravasation mediated by the integrin α5β1, p38 mitogen-activated protein kinase, and matrix metalloproteinases. The tolloid-like 1 (TLL1) protease cleaved ANGPTL2 into fragments in vitro that did not enhance tumor progression when overexpressed in xenografts. Expression of TLL1 was weak in OS patient tumors, suggesting that ANGPTL2 may not be efficiently cleaved upon secretion from OS cells. These findings demonstrate that preventing ANGPTL2 signaling stimulated by the tumor microenvironment could inhibit tumor cell migration and metastasis.

Angiopoietin-like protein 2 accelerates carcinogenesis by activating chronic inflammation and oxidative stress

Chronic inflammation has received much attention as a risk factor for carcinogenesis. We recently reported that Angiopoietin-like protein 2 (Angptl2) facilitates inflammatory carcinogenesis and metastasis in a chemically induced squamous cell carcinoma (SCC) of the skin mouse model. In particular, we demonstrated that Angptl2-induced inflammation enhanced susceptibility of skin tissues to “preneoplastic change” and “malignant conversion” in SCC development; however, mechanisms underlying this activity remain unclear. Using this model, we now report that transgenic mice overexpressing Angptl2 in skin epithelial cells (K14-Angptl2 Tg mice) show enhanced oxidative stress in these tissues. Conversely, in the context of this model, Angptl2 knockout (KO) mice show significantly decreased oxidative stress in skin tissue as well as a lower incidence of SCC compared with wild-type mice. In the chemically induced SCC model, treatment of K14-Angptl2 Tg mice with the antioxidant N-acetyl cysteine (NAC) significantly reduced oxidative stress in skin tissue and the frequency of SCC development. Interestingly, K14-Angptl2 Tg mice in the model also showed significantly decreased expression of mRNA encoding the DNA mismatch repair enzyme Msh2 compared with wild-type mice and increased methylation of the Msh2 promoter in skin tissues. Msh2 expression in skin tissues of Tg mice was significantly increased by NAC treatment, as was Msh2 promoter demethylation. Overall, this study strongly suggests that the inflammatory mediator Angptl2 accelerates chemically induced carcinogenesis through increased oxidative stress and decreased Msh2 expression in skin tissue. Implications: Angptl2-induced inflammation increases susceptibility to microenvironmental changes, allowing increased oxidative stress and decreased Msh2 expression; therefore, Angptl2 might be a target to develop new strategies to antagonize these activities in premalignant tissue. Mol Cancer Res; 12(2); 239–49. ©2013 AACR.

Angiopoietin-like protein 2 induced by mechanical stress accelerates degeneration and hypertrophy of the ligamentum flavum in lumbar spinal canal stenosis

Chronic inflammation and subsequent fibrosis induced by mechanical stress play an important role in ligamentum flavum (LF) hypertrophy and degeneration in patients with lumbar spinal canal stenosis (LSCS). Angiopoietin-like protein 2 (Angptl2) is a chronic inflammatory mediator induced under various pathological conditions and increases the expression of TGF-β1, which is a well-characterized mediator in LF hypertrophy. We investigated whether Angptl2 is induced by mechanical stress, and whether it contributes to LF hypertrophy and degeneration by activating the TGF-β1 signaling cascade. In this study, we investigated human LF tissue and LF fibroblasts isolated from patients who underwent lumbar surgery. We found that Angptl2 was abundantly expressed in fibroblasts of hypertrophied LF tissues at both the mRNA and protein levels. This expression was not only positively correlated with LF thickness and degeneration but also positively correlated with lumbar segmental motion. Our in vitro experiments with fibroblasts from hypertrophied LF tissue revealed that mechanical stretching stress increases the expression and secretion of Angptl2 via activation of calcineurin/NFAT pathways. In hypertrophied LF tissue, expression of TGF-β1 mRNA was also increased and TGF-β1/Smad signaling was activated. Angptl2 expression in LF tissue was positively correlated with the expression of TGF-β1 mRNA, suggesting cooperation between Angptl2 and TGF-β1 in the pathogenesis of LF hypertrophy. In vitro experiments revealed that Angptl2 increased levels of TGF-β1 and its receptors, and also activated TGF-β1/Smad signaling. Mechanical stretching stress increased TGF-β1 mRNA expression, which was partially attenuated by treatment with a calcineurin/NFAT inhibitor or Angptl2 siRNA, indicating that induction of TGF-β1 expression by mechanical stretching stress is partially mediated by Angptl2. We conclude that expression of Angptl2 induced by mechanical stress in LF fibroblasts promotes LF tissue degeneration by activation of TGF-β1/Smad signaling, which results in LF hypertrophy in patients with LSCS.

Serum ANGPTL2 levels reflect clinical features of breast cancer patients: implications for the pathogenesis of breast cancer metastasis

Introduction Breast cancer is a leading cause of cancer-related death in women worldwide, and its metastasis is a major cause of disease mortality. Therefore, identification of the mechanisms underlying breast cancer metastasis is crucial for the development of therapeutic and diagnostic strategies. Our recent study of immunodeficient female mice transplanted with MDA-MB231 breast cancer cells demonstrated that tumor cell-derived angiopoietin-like protein 2 (ANGPTL2) accelerates metastasis through both increasing tumor cell migration in an autocrine/paracrine manner, and enhancing tumor angiogenesis. To determine whether ANGPTL2 contributes to its clinical pathogenesis, we asked whether serum ANGPTL2 levels reflect the clinical features of breast cancer progression. Methods We monitored the levels of secreted ANGPTL2 in supernatants of cultured proliferating MDA-MB231 cells. We also determined whether the circulating ANGPTL2 levels were positively correlated with cancer progression in an in vivo breast cancer xenograft model using MDA-MB231 cells. Finally, we investigated whether serum ANGPTL2 levels were associated with clinical features in breast cancer patients. Results Both in vitro and in vivo experiments showed that the levels of ANGPTL2 secreted from breast cancer cells increased with cell proliferation and cancer progression. Serum ANGPTL2 levels in patients with metastatic breast cancer were significantly higher than those in healthy subjects or in patients with ductal carcinoma in situ or non-metastatic invasive ductal carcinoma. Serum ANGPTL2 levels in patients negative for estrogen receptors and progesterone receptors, particularly triple-negative cases, reflected histological grades. Conclusions These findings suggest that serum ANGPTL2 levels in breast cancer patients could represent a potential marker of breast cancer metastasis.

ANGPTL2 increases bone metastasis of breast cancer cells through enhancing CXCR4 signaling

Bone metastasis of breast cancer cells is a major concern, as it causes increased morbidity and mortality in patients. Bone tissue-derived CXCL12 preferentially recruits breast cancer cells expressing CXCR4 to bone metastatic sites. Thus, understanding how CXCR4 expression is regulated in breast cancer cells could suggest approaches to decrease bone metastasis of breast tumor cells. Here, we show that tumor cell-derived angiopoietin-like protein 2 (ANGPTL2) increases responsiveness of breast cancer cells to CXCL12 by promoting up-regulation of CXCR4 in those cells. In addition, we used a xenograft mouse model established by intracardiac injection of tumor cells to show that ANGPTL2 knockdown in breast cancer cells attenuates tumor cell responsiveness to CXCL12 by decreasing CXCR4 expression in those cells, thereby decreasing bone metastasis. Finally, we found that ANGPTL2 and CXCR4 expression levels within primary tumor tissues from breast cancer patients are positively correlated. We conclude that tumor cell-derived ANGPTL2 may increase bone metastasis by enhancing breast tumor cell responsiveness to CXCL12 signaling through up-regulation of tumor cell CXCR4 expression. These findings may suggest novel therapeutic approaches to treat metastatic breast cancer.

Angiopoietin-like protein 2 renders colorectal cancer cells resistant to chemotherapy by activating spleen tyrosine kinase–phosphoinositide 3-kinase-dependent anti-apoptotic signaling

Angiopoietin‐like protein 2 (ANGPTL2) plays an important role in inflammatory carcinogenesis and tumor metastasis by activating tumor angiogenesis and tumor cell chemotaxis and invasiveness. However, it is unclear whether ANGPTL2 expression has an effect on tumor cell survival. Here, we explored that possibility by determining whether ANGPTL2 expression altered survival of human colorectal cancer cell lines treated with antineoplastic drugs. To do so, we generated SW480 cells expressing ANGPTL2 (SW480/ANGPTL2) and control (SW480/Ctrl) cells. Apoptosis induced by antineoplastic drug treatment was significantly decreased in SW480/ANGPTL2 compared to control cells. Expression of anti‐apoptotic BCL‐2 family genes was upregulated in SW480/ANGPTL2 compared to SW480/Ctrl cells. To assess signaling downstream of ANGPTL2 underlying this effect, we carried out RNA sequencing analysis of SW480/ANGPTL2 and SW480/Ctrl cells. That analysis, combined with in vitro experiments, indicated that Syk‐PI3K signaling induced expression of BCL‐2 family genes in SW480/ANGPTL2 cells. Furthermore, ANGPTL2 increased its own expression in a feedback loop by activating the spleen tyrosine kinase–nuclear factor of activated T cells (Syk–NFAT) pathway. Finally, we observed a correlation between higher ANGPTL2 expression in primary unresectable tumors from colorectal cancer patients who underwent chemotherapy with a lower objective response rate. These findings suggest that attenuating ANGPTL2 signaling in tumor cells may block tumor cell resistance to antineoplastic therapies.

Mice Deficient in Angiopoietin-like Protein 2 (Angptl2) Gene Show Increased Susceptibility to Bacterial Infection Due to Attenuated Macrophage Activity

Macrophages play crucial roles in combatting infectious disease by promoting inflammation and phagocytosis. Angiopoietin-like protein 2 (ANGPTL2) is a secreted factor that induces tissue inflammation by attracting and activating macrophages to produce inflammatory cytokines in chronic inflammation-associated diseases such as obesity-associated metabolic syndrome, atherosclerosis, and rheumatoid arthritis. Here, we asked whether and how ANGPTL2 activates macrophages in the innate immune response. ANGPTL2 was predominantly expressed in proinflammatory mouse bone marrow-derived differentiated macrophages (GM-BMMs) following GM-CSF treatment relative to anti-inflammatory cells (M-BMMs) established by M-CSF treatment. Expression of the proinflammatory markers IL-1β, IL-12p35, and IL-12p40 significantly decreased in GM-BMMs from Angptl2-deficient compared with wild-type (WT) mice, suggestive of attenuated proinflammatory activity. We also report that ANGPTL2 inflammatory signaling is transduced through integrin α5β1 rather than through paired immunoglobulin-like receptor B. Interestingly, Angptl2-deficient mice were more susceptible to infection with Salmonella enterica serovar Typhimurium than were WT mice. Moreover, nitric oxide (NO) production by Angptl2-deficient GM-BMMs was significantly lower than in WT GM-BMMs. Collectively, our findings suggest that macrophage-derived ANGPTL2 promotes an innate immune response in those cells by enhancing proinflammatory activity and NO production required to fight infection.

Corrigendum: ANGPTL2 increases bone metastasis of breast cancer cells through enhancing CXCR4 signaling.

Corrigendum: ANGPTL2 increases bone metastasis of breast cancer cells through enhancing CXCR4 signaling