Inactivation of axon guidance molecule netrin-1 in human colorectal cancer by an epigenetic mechanism.

Netrin-1, the protein product of the NTN1 gene, is an axon guidance molecule implicated in regulation of cell survival and tumorigenesis. Expression of the netrin-1 receptors deleted in colorectal cancer (DCC) and uncoordinated 5 homolog (UNC5H) is frequently silenced in colorectal cancer (CRC) by either loss of heterozygosity or epigenetic mechanisms. However, netrin-1 expression and regulation in CRC are mostly unknown. Here, we report that NTN1 expression is significantly reduced in most CRC tissues compared to the adjacent normal intestinal mucosa, and that NTN1 DNA methylation is significantly higher in CRCs (24.6%) than in the adjacent normal intestinal mucosa (4.0%). In 6 CRC cell lines, NTN1 expression is low. Treatment with 5-Aza-2'-deoxycytidine increased expression of NTN1 in CRC cell lines, indicating that DNA methylation represses NTN1 transcription in CRCs. NTN1 DNA hypermethylation was significantly associated with advanced CRC disease. Median netrin-1 serum levels were significantly decreased in CRC patients (330.1 pg/mL) compared with normal individuals (438.6 pg/mL). Our results suggest that netrin-1 is a candidate biomarker for CRC.

The Exocyst: Dynamic Machine or Static Tethering Complex?

The exocyst is a conserved octameric complex that physically tethers a vesicle to the plasma membrane, prior to membrane fusion. It is important not only for secretion and membrane delivery but also, in mammalian cells, for cytokinesis, ciliogenesis, autophagy, tumorigenesis, and host defense. The combination of genome editing and advanced light microscopy of exocyst subunits in living cells has recently shown the complex to be much more dynamic than previously appreciated, and exposed how little we still know about its function and regulation.

Ectodomain Shedding of Lymphatic Vessel Endothelial Hyaluronan Receptor 1 (LYVE-1) Is Induced by Vascular Endothelial Growth Factor A (VEGF-A).

Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), a type I transmembrane glycoprotein, is known as one of the most specific lymphatic vessel markers in the skin. In this study, we found that the ectodomain of LYVE-1 undergoes proteolytic cleavage, and this process produces soluble LYVE-1. We further identified the cleavage site for ectodomain shedding and generated an uncleavable mutant of LYVE-1. In lymphatic endothelial cells, ectodomain shedding of LYVE-1 was induced by vascular endothelial growth factor (VEGF)-A, an important factor for angiogenesis and lymphangiogenesis under pathological conditions. VEGF-A-induced LYVE-1 ectodomain shedding was mediated via the extracellular signal-regulated kinase (ERK) and a disintegrin and metalloproteinase (ADAM) 17. Wild-type LYVE-1, but not uncleavable LYVE-1, promoted migration of lymphatic endothelial cells in response to VEGF-A. Immunostaining analyses in human psoriasis skin lesions and VEGF-A transgenic mouse skin suggested that the ectodomain shedding of LYVE-1 occurred in lymphatic vessels undergoing chronic inflammation. These results indicate that the ectodomain shedding of LYVE-1 might be involved in promoting pathological lymphangiogenesis.

Human antigen R-mediated mRNA stabilization is required for ultraviolet B-induced autoinduction of amphiregulin in keratinocytes.

All members of the EGF family are produced as transmembrane precursors that are proteolytically processed into soluble forms by disintegrin and metalloproteinases (ADAMs) for autocrine/paracrine pathways. In turn, the ligand-activated EGF receptor (EGFR) induces the expression of EGF family members, so-called "autoinduction." However, it is not well understood how this autoinduction occurs. In this study, we investigated the molecular mechanism of the autoinduction of amphiregulin (AREG), a member of the EGF family. We found that ultraviolet B (UVB) exposure increased the AREG mRNA level by stabilization of its mRNA in a human immortalized keratinocyte cell line, HaCaT. The 3' UTR of AREG mRNA was responsible for binding to an mRNA-binding protein, human antigen R (HuR), and the interaction between AREG mRNA and HuR was enhanced by UVB. Inducible knockdown of HuR expression significantly decreased AREG mRNA stability. Interestingly, treatment of HaCaT cells with an EGFR inhibitor, an EGFR neutralizing antibody, or an ADAM inhibitor destabilized AREG mRNA. In the case of ADAM inhibition, administration of soluble AREG restored the mRNA level, indicating that the stabilization occurs in a shedding-dependent manner of EGFR ligands. The HuR dependence of AREG mRNA and protein expression was also confirmed in human primary keratinocytes. Taken together, we propose a novel mechanism by which HuR regulates the stability of AREG mRNA in keratinocytes after UVB exposure and suggest that targeting of HuR functions might be crucial for understanding skin cancers caused by aberrant EGF family member-EGFR signaling.

Monoubiquitination of pro-amphiregulin regulates its endocytosis and ectodomain shedding.

All members of epidermal growth factor (EGF) family are expressed as transmembrane precursors on cell surfaces and then proteolytically converted to soluble ligands for EGF receptor (EGFR) by a disintegrin and metalloproteases (ADAMs). As enzyme-substrate complex formation is essential for this "ectodomain shedding", alteration of cell surface retention could affect their physical interaction with ADAMs and eventually contribute to shedding efficiency. Here, we showed that monoubiquitination of pro-amphiregulin (pro-AREG, an EGFR ligand) accelerated its half-life on cell surface. Monoubiquitination occurred at lysine 240 of pro-AREG as the primary acceptor site. Using a chimeric protein of pro-AREG and a monomeric ubiquitin mutant (pro-AREGmUb), immunocytochemical analysis and a cell surface biotinylation assay revealed that a significant portion of pro-AREGmUb was expressed on the cell surface, immediately endocytosed, and predominantly localized to early endosomes. Importantly, ectodomain shedding of pro-AREGmUb induced by tetradecanoyl phorbol acetate was significantly reduced in comparison to wild-type pro-AREG. These results suggested that pro-AREG monoubiquitination and the subsequent trafficking to intracellular organelles is a novel shedding regulatory mechanism that contributes to the secretion of EGFR ligands in growth factor signaling.

Inhibitory Effect of the HASPIN Inhibitor CHR-6494 on BxPC-3-Luc, A Luciferase-Expressing Pancreatic Cancer Cell Line.

HASPIN acts in chromosome segregation via histone phosphorylation. Recently, HASPIN inhibitors have been shown to suppress growth of various cancer cells. Pancreatic cancer has no symptom in the early stages and may progress before detection. So, the 5-year survival rate is low. Here, we reported that administration of the HASPIN inhibitor, CHR-6494, to mice bearing pancreatic BxPC-3-Luc cancer cells significantly suppressed growth of BxPC-3-Luc cells. CHR-6494 might be a useful agent for treating pancreatic cancer.

Evaluation of the antiproliferative effects of the HASPIN inhibitor CHR-6494 in breast cancer cell lines.

HASPIN is a serine/threonine kinase that regulates mitosis by phosphorylating histone H3 at threonine 3. The expression levels of HASPIN in various cancers are associated with tumor malignancy and poor survival, suggesting that HASPIN inhibition may suppress cancer growth. As HASPIN mRNA levels are elevated in human breast cancer tissues compared with adjacent normal tissues, we examined the growth-suppressive effects of CHR-6494, a potent HASPIN inhibitor, in breast cancer cell lines in vitro and in vivo. We found that HASPIN was expressed in breast cancer cells of all molecular subtypes, as well as in immortalized mammary epithelial cells. HASPIN expression levels appeared to be correlated with the cell growth rate but not the molecular subtype of breast cancer. CHR-6494 exhibited potent antiproliferative effects against breast cancer cell lines and immortalized mammary epithelial cells in vitro, but failed to inhibit the growth of MDA-MB-231 xenografted tumors under conditions that have significant effects in a colorectal cancer model. These results imply that CHR-6494 does have antiproliferative effects in some situations, and further drug screening efforts are anticipated to identify more potent and selective HASPIN inhibition for use as an anticancer agent in breast cancer patients.

Nodal lymphangiogenesis and metastasis: Role of tumor-induced lymphatic vessel activation in extramammary Paget's disease.

Nodal lymphangiogenesis promotes distant lymph node (LN) metastasis in experimental cancer models. However, the role of nodal lymphangiogenesis in distant metastasis and in the overall survival of cancer patients remains unknown. Therefore, we investigated mechanisms that might facilitate regional and distant LN metastasis in extramammary Paget's disease (EMPD). We retrospectively analyzed the impact of tumor-induced lymphatic vessel activation on the survival of 116 patients, the largest cohort with EMPD studied to date. Nodal lymphangiogenesis was significantly increased in metastatic, compared with tumor-free, LNs (P = 0.022). Increased lymphatic invasion within regional LNs was significantly associated with distant metastasis in LN (P = 0.047) and organs (P = 0.003). Thus, invasion within regional LNs is a powerful indicator of systemic tumor spread and reduced patient survival in EMPD (P = 0.0004). Lymphatic vessels associated with tumors expressed stromal cell-derived factor-1 (SDF-1), whereas CXCR4 was expressed on invasive Paget cells undergoing epithelial-mesenchymal transition (EMT)-like process. A431 cells overexpressing Snail expressed increased levels of CXCR4 in the presence of transforming growth factor-beta1. Haptotactic migration assays confirmed that Snail-induced EMT-like process promotes tumor cell motility via the CXCR4-SDF-1 axis. Sinusoidal lymphatic endothelial cells and macrophages expressed SDF-1 in subcapsular sinuses of lymph nodes before Paget cell arrival. Our findings reveal that EMT-related features likely promote lymphatic metastasis of EMPD by activating the CXCR4-SDF-1 axis.

An IMiD-induced SALL4 degron system for selective degradation of target proteins.

Regulating the amount of proteins in living cells is a powerful approach for understanding the functions of the proteins. Immunomodulatory drugs (IMiDs) induce the degradation of neosubstrates by interacting with celebron (CRBN) in the cullin E3 ubiquitin ligase complex (CRL4CRBN). Here, we developed the IMiD-dependent Sal-like protein 4 (SALL4) degron (S4D) system for chemical protein knockdown. In transient assays, an N- or C-terminal S4D tag induced the degradation of proteins localized to various subcellular compartments, including the plasma membrane. The activity of luciferase-S4D was reduced by 90% within 3 h of IMiD treatment. IMiD treatment reduced the expression of endogenous S4D-fused RelA and IκBα in knock-in (KI) experiments. Interestingly, the IκBα knockdown suggested that there may be another, unknown mechanism for RelA translocation to the nucleus. Furthermore, 5-hydroxythalidomide as a thalidomide metabolite specifically degradated S4D-tagged protein. These results indicate that the S4D system is a useful tool for cellular biology.

Publisher Correction: Exocyst dynamics during vesicle tethering and fusion.

The original version of this Article contained errors in the author affiliations. Affiliation 2 incorrectly read 'Department of Biochemistry and Molecular Genetics and Breast Surgery, Ehime University Graduate School of Medicine, 7910295, Japan' and affiliation 3 incorrectly read 'Department of Hepato-Biliary-Pancreatic and Breast Surgery, Ehime University Graduate School of Medicine, Matsuyama 7910295, Japan.' These errors have now been corrected in both the PDF and HTML versions of the Article.

Exocyst dynamics during vesicle tethering and fusion.

The exocyst is a conserved octameric complex that tethers exocytic vesicles to the plasma membrane prior to fusion. Exocyst assembly and delivery mechanisms remain unclear, especially in mammalian cells. Here we tagged multiple endogenous exocyst subunits with sfGFP or Halo using Cas9 gene-editing, to create single and double knock-in lines of mammary epithelial cells, and interrogated exocyst dynamics by high-speed imaging and correlation spectroscopy. We discovered that mammalian exocyst is comprised of tetrameric subcomplexes that can associate independently with vesicles and plasma membrane and are in dynamic equilibrium with octamer and monomers. Membrane arrival times are similar for subunits and vesicles, but with a small delay (~80msec) between subcomplexes. Departure of SEC3 occurs prior to fusion, whereas other subunits depart just after fusion. About 9 exocyst complexes are associated per vesicle. These data reveal the mammalian exocyst as a remarkably dynamic two-part complex and provide important insights into assembly/disassembly mechanisms.

Reversible interconversion and maintenance of mammary epithelial cell characteristics by the ligand-regulated EGFR system.

Epithelial cell plasticity is controlled by extracellular cues, but the underlying mechanisms remain to be fully understood. Epidermal growth factor (EGF) and amphiregulin (AREG) are high- and low-affinity ligands for EGF receptor (EGFR), respectively. EGFR signaling is known to promote epithelial-mesenchymal transition (EMT) by the activation of ERK and the induction of an EMT transcription factor, ZEB1. Here, we demonstrate that ligand-switching between EGF and AREG at equivalent molarity reversibly interconverts epithelial and mesenchymal-like states of EGFR signal-dependent mammary epithelial cells. The EGF- and AREG-cultured cells also differ in their epithelial characteristics, including the expression of cell surface markers, the mode of migration and the ability for acinus-formation. The ligand-switching between EGF and AREG temporally alters strength of the shared EGFR-ERK signaling. This alteration inverts relative expression levels of ZEB1 and its antagonizing microRNAs, miR-205 and miR-200c, those are critical determinants of the epithelial phenotype. Further, AREG-induced EGFR accumulation on the plasma membrane compensates for the weak association between AREG and EGFR. The EGFR dynamics enables AREG to support proliferation as efficiently as EGF at equivalent molarity and to maintain epithelial characteristics. Our findings reveal a role of EGFR ligands-generated signal strength in the regulation of mammary epithelial cell plasticity.

Cell surface annexins regulate ADAM-mediated ectodomain shedding of proamphiregulin.

A disintegrin and metalloproteinase (ADAM) is a family of enzymes involved in ectodomain shedding of various membrane proteins. However, the molecular mechanism underlying substrate recognition by ADAMs remains unknown. In this study, we successfully captured and analyzed cell surface transient assemblies between the transmembrane amphiregulin precursor (proAREG) and ADAM17 during an early shedding phase, which enabled the identification of cell surface annexins as components of their shedding complex. Annexin family members annexin A2 (ANXA2), A8, and A9 interacted with proAREG and ADAM17 on the cell surface. Shedding of proAREG was increased when ANXA2 was knocked down but decreased with ANXA8 and A9 knockdown, because of enhanced and impaired association with ADAM17, respectively. Knockdown of ANXA2 and A8 in primary keratinocytes altered wound-induced cell migration and ultraviolet B-induced phosphorylation of epidermal growth factor receptor (EGFR), suggesting that annexins play an essential role in the ADAM-mediated ectodomain shedding of EGFR ligands. On the basis of these data, we propose that annexins on the cell surface function as "shedding platform" proteins to determine the substrate selectivity of ADAM17, with possible therapeutic potential in ADAM-related diseases.

Essential roles of androgen signaling in Wolffian duct stabilization and epididymal cell differentiation.

The epididymis is a male accessory organ and functions for sperm maturation and storage under the control of androgen. The development of the epididymis is also androgen dependent. The Wolffian duct (WD), anlagen of the epididymis, is formed in both male and female embryos; however, it is stabilized only in male embryos by testicular androgen. Androgen drives subsequent differentiation of the WD into the epididymis. Although the essential roles of androgen in WD masculinization and epididymal function have been established, little is known about cellular events regulated precisely by androgen signaling during these processes. It is also unclear whether androgen signaling, especially in the epithelia, has further function for epididymal epithelial cell differentiation. In this study we examined the cellular death and proliferation controlled by androgen signaling via the androgen receptor (AR) in WD stabilization. Analyses using AR knockout mice revealed that androgen signaling inhibits epithelial cell death in this process. Analysis of AP2α-Cre;AR(flox/Y) mice, in which AR function is deleted in the WD epithelium, revealed that epithelial AR is not required for the WD stabilization but is required for epithelial cell differentiation in the epididymis. Specifically, loss of epithelial AR significantly reduced expression of p63 that is essential for differentiation of basal cells in the epididymal epithelium. We also interrogated the possibility of regulation of the p63 gene (Trp63) by AR in vitro and found that p63 is a likely direct target of AR regulation.