SAP deletion promotes malignant insulinoma progression by inducing CXCL12 secretion from CAFs via the CXCR4/p38/ERK signalling pathway.

Malignant insulinoma is an extremely rare type of functioning pancreatic neuroendocrine tumour with a high degree of malignancy and a high incidence of metastasis. However, it is still unclear how malignant insulinomas develop and metastasize. Serum amyloid P component (SAP), a member of the pentraxin protein family, is an acute-phase protein secreted by liver cells. The role of SAP in insulinoma and the related mechanism are still unknown. To determine the effect of SAP on insulinoma, we crossed Rip1-Tag2 mice, which spontaneously develop insulinoma, and SAP knockout (KO) mice to generate Rip1-Tag2;SAP-/- mice. We found that SAP deletion significantly promoted the growth, invasion and metastasis of malignant insulinoma through C-X-C motif chemokine ligand 12 (CXCL12) secreted by cancer-associated fibroblasts (CAFs). Further study showed that SAP deletion promoted CXCL12 secretion by CAFs through the CXCR4/p38/ERK signalling pathway. These findings reveal a novel role and mechanism of SAP in malignant insulinoma and provide direct evidence that SAP may be a therapeutic agent for this disease.

NF-κB Activator 1 downregulation in macrophages activates STAT3 to promote adenoma-adenocarcinoma transition and immunosuppression in colorectal cancer.

BACKGROUND: Adenoma-adenocarcinoma transition is a key feature of colorectal cancer (CRC) occurrence and is closely regulated by tumor-associated macrophages (TAMs) and CD8+ T cells. Here, we investigated the effect of the NF-κB activator 1 (Act1) downregulation of macrophages in the adenoma-adenocarcinoma transition. METHODS: This study used spontaneous adenoma-developing ApcMin/+, macrophage-specific Act1-knockdown (anti-Act1), and ApcMin/+; anti-Act1 (AA) mice. Histological analysis was performed on CRC tissues of patients and mice. CRC patients' data retrieved from the TCGA dataset were analyzed. Primary cell isolation, co-culture system, RNA-seq, and fluorescence-activated cell sorting (FACS) were used. RESULTS: By TCGA and TISIDB analysis, the downregulation of Act1 expression in tumor tissues of CRC patients negatively correlated with accumulated CD68+ macrophages in the tumor. Relative expression of EMT markers in the tumor enriched ACT1lowCD68+ macrophages of CRC patients. AA mice showed adenoma-adenocarcinoma transition, TAMs recruitment, and CD8+ T cell infiltration in the tumor. Macrophages depletion in AA mice reversed adenocarcinoma, reduced tumor amounts, and suppressed CD8+ T cell infiltration. Besides, macrophage depletion or anti-CD8a effectively inhibited metastatic nodules in the lung metastasis mouse model of anti-Act1 mice. CRC cells induced activation of IL-6/STAT3 and IFN-γ/NF-κB signaling and the expressions of CXCL9/10, IL-6, and PD-L1 in anti-Act1 macrophages. Anti-Act1 macrophages facilitated epithelial-mesenchymal-transition and CRC cells' migration via CXCL9/10-CXCR3-axis. Furthermore, anti-Act1 macrophages promoted exhaustive PD1+ Tim3+ CD8+ T cell formation. Anti-PD-L1 treatment repressed adenoma-adenocarcinoma transition in AA mice. Silencing STAT3 in anti-Act1 macrophages reduced CXCL9/10 and PD-L1 expression and correspondingly inhibited epithelial-mesenchymal-transition and CRC cells' migration. CONCLUSIONS: Act1 downregulation in macrophages activates STAT3 that promotes adenoma-adenocarcinoma transition via CXCL9/10-CXCR3-axis in CRC cells and PD-1/PD-L1-axis in CD8+ T cells.

CYD0281, a Bcl-2 BH4 domain antagonist, inhibits tumor angiogenesis and breast cancer tumor growth.

BACKGROUND: B-cell lymphoma 2 (Bcl-2) family proteins are key regulators of apoptosis, which possess four conserved Bcl-2 homologies (BH) domains. Among the BH domains, the BH3 domain is considered as a potent 'death domain' while the BH4 domain is required for anti-apoptotic activity. Bcl-2 can be converted to a pro-apoptotic molecule through the removal or mutation of the BH4 domain. Bcl-2 is considered as an inducer of angiogenesis, which can promote tumor vascular network formation and further afford nutrients and oxygen to promote tumor progression. However, whether disrupting the function of the BH4 domain to convert Bcl-2 into a pro-apoptotic molecule could make Bcl-2 possess the potential for anti-angiogenic therapy remains to be defined. METHODS: CYD0281 was designed and synthesized according to the lead structure of BDA-366, and its function on inducing a conformational change of Bcl-2 was further evaluated via immunoprecipitation (IP) and immunofluorescence (IF) assays. Moreover, the function of CYD0281 on apoptosis of endothelial cells was analyzed via cell viability, flow cytometry, and western blotting assays. Additionally, the role of CYD0281 on angiogenesis in vitro was determined via endothelial cell migration and tube formation assays and rat aortic ring assay. Chick embryo chorioallantoic membrane (CAM) and yolk sac membrane (YSM) models, breast cancer cell xenograft tumor on CAM and in mouse models as well as the Matrigel plug angiogenesis assay were used to explore the effects of CYD0281 on angiogenesis in vivo. RESULTS: We identified a novel potent small-molecule Bcl-2-BH4 domain antagonist, CYD0281, which exhibited significant anti-angiogenic effects both in vitro and in vivo, and further inhibited breast cancer tumor growth. CYD0281 was found to induce conformational changes in Bcl-2 through the exposure of the BH3 domain and convert Bcl-2 from an anti-apoptotic molecule into a cell death inducer, thereby resulting in the apoptosis of vascular endothelial cells. CONCLUSIONS: This study has revealed CYD0281 as a novel Bcl-2-BH4 antagonist that induces conformational changes of Bcl-2 to convert to a pro-apoptotic molecule. Our findings indicate that CYD0281 plays a crucial role in anti-angiogenesis and may be further developed as a potential anti-tumor drug candidate for breast cancer. This work also provides a potential anti-angiogenic strategy for breast cancer treatment.

R5, a neutralizing antibody to Robo1, suppresses breast cancer growth and metastasis by inhibiting angiogenesis via down-regulating filamin A.

Breast cancer (BC) is one of the most common cancers among women in both developed and developing countries with a rising incidence. Using the MMTV-PyMT transgenic mouse model and xenografted breast cancer model, we found that R5, a neutralizing antibody to Robo1, significantly inhibited BC growth and metastasis. Angiogenesis is involved in the growth and metastasis of BC. Interestingly, R5 significantly decreases microvessel density in BC tissues, and inhibits blood vessel formation and development in in vivo chick embryo chorioallantoic membrane (CAM), yolk sac membrane (YSM) and Matrigel plug models. To investigate whether its anti-breast cancer efficacy is ascribed to its direct antiangiogenic properties, xenografted breast cancer model on CAM was established. Furthermore, R5 significantly reduces the tube formation of the vascular plexus on xenografted breast tumor on CAM. R5 also suppresses the migration and the tubular structure formation of human umbilical vein endothelial cells (HUVECs) by down-regulating the expression of filamin A (FLNA). These findings show that R5 has the potential to be a promising agent for the treatment of BC by suppressing the tumor-induced angiogenesis.

Decylubiquinone suppresses breast cancer growth and metastasis by inhibiting angiogenesis via the ROS/p53/ BAI1 signaling pathway.

Breast cancer is one of the most common cancers worldwide with a rising incidence, and is the leading cause of cancer-related death among females. Angiogenesis plays an important role in breast cancer growth and metastasis. In this study, we identify decylubiquinone (DUb), a coenzyme Q10 analog, as a promising anti-breast cancer agent through suppressing tumor-induced angiogenesis. We screened a library comprising FDA-approved drugs and found that DUb significantly inhibits blood vessel formation using in vivo chick embryo chorioallantoic membrane (CAM) and yolk sac membrane (YSM) models. DUb was further identified to inhibit angiogenesis in the rat aortic ring and Matrigel plug assay. Moreover, DUb was found to suppress breast cancer growth and metastasis in the MMTV-PyMT transgenic mouse and human xenograft tumor models. To explore whether the anticancer efficacy of DUb was directly corrected with tumor-induced angiogenesis, the MDA-MB-231 breast cancer assay on the CAM was performed. Interestingly, DUb significantly inhibits the angiogenesis of breast cancer on the CAM. Brain angiogenesis inhibitor 1 (BAI1), a member of the G protein-coupled receptor (GPCR) adhesion subfamily, has an important effect on the inhibition of angiogenesis. Further studies demonstrate that DUb suppresses the formation of tubular structures by regulating the reactive oxygen species (ROS)/p53/BAI1 signaling pathway. These results uncover a novel finding that DUb has the potential to be an effective agent for the treatment of breast cancer by inhibiting tumor-induced angiogenesis.

Knocking out matrix metalloproteinase 12 causes the accumulation of M2 macrophages in intestinal tumor microenvironment of mice.

MMP12 is mainly secreted by macrophages, is involved in macrophage development, and decomposes the extracellular matrix. Herein, we investigated whether macrophages would change in the intestinal tumor microenvironment after MMP12 knockout. ApcMin/+;MMP12-/-mice were obtained by crossbreeding ApcMin/+ mice with MMP12 knockout mice (MMP12-/- mice). The data showed that the number and volume of intestinal tumors were significantly increased in ApcMin/+;MMP12-/- mice compared with ApcMin/+ mice. Additionally, the tumor biomarkers CA19-9, CEA, and ß-catenin appeared relatively early in intestinal tumors in ApcMin/+;MMP12-/- mice. The results demonstrated that knocking out MMP12 accelerated the tumor growth and pathological process. On further investigation of its mechanism, the proportions of M2 macrophages in the spleen and among peritoneal macrophages were significantly up-regulated in ApcMin/+;MMP12-/- mice. Expression of M2 macrophage-related genes was up-regulated in tumor and peritoneal macrophages. The M2-related cytokine levels of IL-4 and IL-13 were increased in the serum of ApcMin/+;MMP12-/-mice. In vitro, bone marrow-derived M2 macrophages were obtained by treating bone marrow cells with IL-4 and IL-13, and these M2 macrophages secreted cytokines being changed. This finding reveals the crucial role of MMP12 in macrophage development and provides a new target for the control of macrophage polarization. Knocking out MMP12 causes intestinal M2 macrophage accumulation in tumor microenvironment, promoting the growth of intestinal tumors in ApcMin/+ mice.

Inactivation of PI3Kδ induces vascular injury and promotes aneurysm development by upregulating the AP-1/MMP-12 pathway in macrophages.

OBJECTIVE: An aneurysm is an inflammatory vascular condition. Phosphatidylinositol 3-kinases δ is highly expressed in leukocytes, and play a key role in innate immunity. However, the link between phosphatidylinositol 3-kinases δ and aneurysm development has not yet been elucidated. APPROACH AND RESULTS: Carotid ligation unexpectedly induced characteristic aneurysm formation beneath the ligation point in p110δ(D910A/D910A) mice (n=25; P<0.001 versus wild-type). Besides, p110δ inactivation exacerbated CaCl2-induced abdominal aortic aneurysms development. A reverse transcription polymerase chain reaction microarray revealed significant extracellular matrix components degradation and matrix metalloproteinases (MMPs) upregulation in the abdominal aorta of p110δ(D910A/D910A) mice. Similarly, the expression of both collagen I and IV was significantly decreased (n=10; P<0.05 versus wild-type) in carotid artery. Western blot assay confirmed that MMP-12 was significantly upregulated in arteries of p110δ(D910A/D910A) mice (n=10; P<0.01 versus wild-type). In vitro, p110δ inactivation marked increase peritoneal macrophages recruitment and synergistically enhance tumor necrosis factor-α-induced recruitment. A specific phosphatidylinositol 3-kinases δ inhibitor (IC87114) or genetic p110δ inactivation upregulated MMP-12 expression and c-Jun phosphorylation (n=6; P<0.05 versus wild-type macrophages). IC87114 also increased activator protein-1 DNA-binding activity (n=6; P<0.001 versus control) and enhanced the effect of tumor necrosis factor-α on activator protein-1-binding activity (n=5; P<0.01 versus tumor necrosis factor-α treatment groups). Knockdown of c-Jun suppressed the effect of the IC87114 and tumor necrosis factor-α on MMP-12 mRNA expression (n=5 in each group; P<0.01 versus scrRNA treatment groups). CONCLUSIONS: Our findings demonstrate that p110δ inactivation leads to extracellular matrix degradation in vessels and promotes aneurysm development by inducing macrophages migration and upregulating the activator protein-1/MMP-12 pathway in macrophages.

Glipizide suppresses embryonic vasculogenesis and angiogenesis through targeting natriuretic peptide receptor A.

Glipizide, a second-generation sulfonylurea, has been widely used for the treatment of type 2 diabetes. However, it is controversial whether or not glipizide would affect angiogenesis or vasculogenesis. In the present study, we used early chick embryo model to investigate the effect of glipizide on angiogenesis and vasculogenesis, which are the two major processes for embryonic vasculature formation as well as tumor neovascularization. We found that Glipizide suppressed both angiogenesis in yolk-sac membrane (YSM) and blood island formation during developmental vasculogenesis. Glipizide did not affect either the process of epithelial to mesenchymal transition (EMT) or mesoderm cell migration. In addition, it did not interfere with separation of smooth muscle cell progenitors from hemangioblasts. Moreover, natriuretic peptide receptor A (NPRA) has been identified as the putative target for glipizide׳s inhibitory effect on vasculogenesis. When NPRA was overexpressed or activated, blood island formation was reduced. NPRA signaling may play a crucial role in the effect of glipizide on vasculogenesis during early embryonic development.

Activation of Slit2-Robo1 signaling promotes liver fibrosis.

BACKGROUND & AIMS: The secretory protein Slit2 and its receptor Robo1 are believed to regulate cell growth and migration. Here, we aimed to determine whether Slit2-Robo1 signaling mediates the pathogenesis of liver fibrosis. METHODS: Serum levels of Slit2 in patients with liver fibrosis were determined by ELISA. Liver fibrosis was induced in wild-type (WT), Slit2 transgenic (Slit2-Tg) and Robo1(+/-)Robo2(+/-) double heterozygotes (Robo1/2(+/-)) mice by carbon tetrachloride (CCl4). The functional contributions of Slit2-Robo1 signaling in liver fibrosis and activation of hepatic stellate cells (HSCs) were investigated using primary mouse HSCs and human HSC cell line LX-2. RESULTS: Significantly increased serum Slit2 levels and hepatic expression of Slit2 and Robo1 were observed in patients with liver fibrosis. Compared to WT mice, Slit2-Tg mice were much more vulnerable to CCl4-induced liver injury and more readily develop liver fibrosis. Development of hepatic fibrosis in Slit2-Tg mice was associated with a stronger hepatic expression of collagen I and α-smooth muscle actin (α-SMA). However, liver injury and hepatic expression of collagen I and α-SMA were attenuated in CCl4-treated Robo1/2(+/-) mice in response to CCl4 exposure. In vitro, Robo1 neutralizing antibody R5 and Robo1 siRNA downregulated phosphorylation of Smad2, Smad3, PI3K, and AKT in HSCs independent of TGF-ß1. R5 and Robo1 siRNA also inhibited the expression of α-SMA by HSCs. Finally, the protective effect of R5 on the CCl4-induced liver injury and fibrosis was further verified in mice. CONCLUSIONS: Slit2-Robo1 signaling promotes liver injury and fibrosis through activation of HSCs.

Slit2 promotes tumor growth and invasion in chemically induced skin carcinogenesis.

Slit, a neuronal guidance cue, binds to Roundabout (Robo) receptors to modulate neuronal, leukocytic, and endothelial migration. Slit has been reported to have an important effect on tumor growth and metastasis. In the current study, we evaluated the role of Slit2 in skin tumor growth and invasion in mice using a two-step chemical carcinogenesis protocol. We found that Slit2 expression correlated with the loss of basement membrane in the samples of human skin squamous cell carcinoma at different stages of disease progression. Slit2-Tg mice developed significantly more skin tumors than wild-type mice. Furthermore, the skin tumors that occurred in Slit2-Tg mice were significantly larger than those in the wild-type mice 10 weeks after 7,12-dimethylbenz[a]anthracene initiation until the end of the experiment. We also found that pathological development of the wild-type mice was delayed compared with that of Slit2-Tg mice. To further investigate the mechanism of increasing tumors in Slit2-Tg mice, we analyzed the expression of 5-bromo-2'-deoxyuridine (BrdU) in mouse skin lesions and found that the number of BrdU-positive cells and microvessel density in skin lesions were significantly higher in Slit2-Tg mice than in wild-type mice. Histological staining of PAS and type IV collagen and the colocalization of Slit2 and type IV collagen demonstrated varying degrees of loss of the basement membrane in the skin lesions from Slit2-Tg mice that were at the stage of carcinoma in situ. However, the basement membrane was well defined in the wild-type mice. In addition, MMP2, but not MMP9, was upregulated in the skin tissue of Slit2-Tg mice. Interruption of Slit2-Robo1 signaling by the antibody R5 significantly repressed the invasive capability of the squamous cell carcinoma cell line A431. Taken together, our findings reveal that Slit2 promotes DMBA/TPA-induced skin tumorigenesis by increasing cell proliferation, microvessel density, and invasive behavior of cutaneous squamous cell carcinoma, along with loss of basement membrane, by upregulation of MMP2 expression.