Cancer-derived exosomes are considered a major driver of cancer-induced pre-metastatic niche formation at foreign sites, but the mechanisms remain unclear. Here, we show that miR-25-3p, a metastasis-promoting miRNA of colorectal cancer (CRC), can be transferred from CRC cells to endothelial cells via exosomes. Exosomal miR-25-3p regulates the expression of VEGFR2, ZO-1, occludin and Claudin5 in endothelial cells by targeting KLF2 and KLF4, consequently promotes vascular permeability and angiogenesis. In addition, exosomal miR-25-3p from CRC cells dramatically induces vascular leakiness and enhances CRC metastasis in liver and lung of mice. Moreover, the expression level of miR-25-3p from circulating exosomes is significantly higher in CRC patients with metastasis than those without metastasis. Our work suggests that exosomal miR-25-3p is involved in pre-metastatic niche formation and may be used as a blood-based biomarker for CRC metastasis.

G protein-coupled receptors (GPCRs) play pivotal roles in regulating various cellular functions. Although many GPCRs induce NF-B activation, the molecular mechanism of GPCR-induced NF-B activation remains largely unknown. CARMA3 (CARD and MAGUK domain-containing protein 3) is a scaffold molecule with unknown biological functions. By generating CARMA3 knockout mice using the gene targeting approach, here we show CARMA3 is required for GPCR-induced NF-B activation. Mechanistically, we found that CARMA3 deficiency impairs GPCR-induced IB kinase (IKK) activation, although it does not affect GPCR-induced IKK␣/␤ phosphorylation, indicating that inducible phosphorylation of IKK␣/␤ alone is not sufficient to induce its kinase activity. We also found that CARMA3 is physically associated with NEMO/IKK␥, and induces polyubiquitination of an unknown protein(s) that associates with NEMO, likely by linking NEMO to TRAF6. Consistently, we found TRAF6 deficiency also abrogates GPCR-induced NF-B activation. Together, our results provide the genetic evidence that CARMA3 is required for GPCR-induced NF-B activation.[Keywords: NF-B; GPCR; CARMA3; neural tube] Supplemental material is available at http://www.genesdev.org.

Background Centromere Protein F (CENPF) associates with the centromere–kinetochore complex and influences cell proliferation and metastasis in several cancers. The role of CENPF in breast cancer (BC) bone metastasis remains unclear. Methods Using the ONCOMINE database, we compared the expression of CENPF in breast cancer and normal tissues. Findings were confirmed in 60 BC patients through immunohistochemical (IHC) staining. Microarray data from GEO and Kaplan–Meier plots were used analyze the overall survival (OS) and relapse free survival (RFS). Using the GEO databases, we compared the expression of CENPF in primary lesions, lung metastasis lesions and bone metastasis lesions, and validated our findings in BALB/C mouse 4T1 BC models. Based on gene set enrichment analysis (GSEA) and western blot, we predicted the mechanisms by which CENPF regulates BC bone metastasis. Results The ONCOMINE database and immunohistochemical (IHC) showed higher CENPF expression in BC tissue compared to normal tissue. Kaplan–Meier plots also revealed that high CENPF mRNA expression correlated to poor survival and shorter progression-free survival (RFS). From BALB/C mice 4T1 BC models and the GEO database, CENPF was overexpressed in primary lesions, other target organs, and in bone metastasis. Based on gene set enrichment analysis (GSEA) and western blot, we predicted that CENPF regulates the secretion of parathyroid hormone-related peptide (PTHrP) through its ability to activate PI3K–AKT–mTORC1. Conclusion CENPF promotes BC bone metastasis by activating PI3K–AKT–mTORC1 signaling and represents a novel therapeutic target for BC treatment.

Lysophosphatidic acid (LPA) is a bioactive phospholipid and binds to its receptors, a family of G protein-coupled receptors (GPCR), which initiates multiple signaling cascades and leads to activation of several transcription factors, including NF-B. Although LPA-induced signaling pathways have been intensively investigated, the molecular mechanism by which LPA activates NF-B is not fully defined. In this work, we found that ␤-arrestin 2, but not ␤-arrestin 1, is required for LPA-induced NF-B activation and interlukin-6 expression. Mechanistically, we found that ␤-arrestin 2 associated with CARMA3, a scaffold protein that plays an essential role in GPCR-induced NF-B activation, suggesting that ␤-arrestin 2 may recruit CARMA3 to LPA receptors. Although ␤-arrestin 2 deficiency did not affect LPA-induced IKK␣/␤ phosphorylation, it impaired LPA-induced IKK kinase activity, which is consistent with our previous findings that CARMA3 is required for IKK␣/␤ activation but not for the inducible phosphorylation of IKK␣/␤. Together, our results provide the genetic evidence that ␤-arrestin 2 serves as a positive regulator in NF-B signaling pathway by connecting CARMA3 to GPCRs.CARMA3 ͉ G protein-coupled receptor ͉ IKK

IIp45 (aka MIIP) is a newly discovered gene whose protein product inhibits cell migration. HDAC6 is a class IIb deacetylase that specifically deacetylates ␣-tubulin, modulates microtubule dynamics, and promotes cell migration. A yeast two-hybrid assay using IIp45 as bait identified HDAC6 protein as a binding partner of IIp45. This physical interaction of the two functionally antagonistic proteins was confirmed by glutathione S-transferase pulldown assay and coimmunoprecipitation assay in human cells. Serial deletion constructs of HDAC6 were used to characterize the interaction of HDAC6 and IIp45, and this analysis found that the two catalytic domains of HDAC6 protein are required for IIp45 binding. We examined the protein expression patterns of IIp45 and HDAC6 in glioma tissues. Elevated protein levels of HDAC6 were found in high grade glioma samples, in contrast to the decreased protein expression of IIp45. The potential negative regulation of HDAC6 expression by IIp45 was confirmed in cell lines with altered IIp45 expression by constitutive overexpression or small interfering RNA knockdown. Protein turnover study revealed that overexpression of IIp45 significantly reduces the intracellular protein stability of endogenous HDAC6, indicating a possible mechanism for the negative regulation of HDAC6 by IIp45. Results from the HDAC activity assay demonstrated that overexpressed IIp45 effectively decreases HDAC6 activity, increases acetylated ␣-tubulin, and reduces cell migration. The increased cell migration resulting from siIIp45 knockdown was significantly reversed by co-transfection of siHDAC6. Thus, we report here for the first time a novel mechanism by which IIp45 inhibits cell motility through inhibition of HDAC6.Cell motility is important for normal tissue development and remodeling as well as for pathological conditions such as cancer invasion and metastasis (1, 2). Cell motility is controlled by well orchestrated programs that modulate the microtubule stabilization and destabilization cycles that permit cells to move efficiently (3). One of the key regulatory mechanisms of microtubule dynamics is acetylation of ␣-tubulin (4). Acetylation of protein lysine residues is a well recognized post-translational modification that modulates various pathways of cellular signal transduction. Protein acetylation is regulated by a number of acetyltransferases and deacetylases, which include the histone deacetylase (HDAC) 2 family (5). Among the 18 HDAC family members that have been reported in humans, HDAC6, classified as a type II histone deacetylase, is the only member that has two functional deacetylase domains and a zinc finger motif (6). Both catalytic domains are required for HDAC6 enzymatic activity (7). HDAC6 is also unique in that it is localized mainly in the cytoplasm and specifically deacetylates ␣-tubulin, the most abundant microtubule component, rather than histone proteins (8). In addition to ␣-tubulin, heat shock protein 90 (hsp90) and cortactin are two well recognized substrate proteins for HDAC6-mediat...

No abstract

Tissue ICAM-1 and serum sICAM-1 could indicate the stage of HCC, and the potential of hepatoma cells for invasion and metastasis. They may play an important role in the metastasis cascade.

The interactions between fibroblasts and the extracellular matrix in wound contraction are mainly mediated via integrin signaling. Integrin-linked kinase (ILK) is a key mediator in integrin signal transduction. We investigated the role of ILK in cutaneous wound contraction. We found that ILK was involved in cutaneous wound healing in rats, and ILK and PI3K/AKT inhibitors inhibited wound contraction and re-epithelialization, consequently delaying wound healing in vivo. Further, using in vitro studies, we demonstrated that ILK and PI3K/AKT inhibitors suppressed the contraction of fibroblastpopulated collagen lattices, inhibited fibroblast migration, and interrupted the effect of TGF-β 1 on promoting alpha smooth muscle actin (α-SMA) expression in fibroblasts. When ILK expression was directly blocked by ILK small interfering RNA transfection, the migration and α-SMA expression of normal dermal fibroblasts were significantly suppressed as well. The data suggest that the ILK-PI3K/AKT signaling pathway mediates cutaneous wound contraction by regulating fibroblast migration and differentiation to myofibroblasts.