The EGF-like proteins DLK1 and DLK2 function as inhibitory non-canonical ligands of NOTCH1 receptor that modulate each other's activities

The protein DLK2, highly homologous to DLK1, belongs to the EGF-like family of membrane proteins, which includes NOTCH receptors and their DSL-ligands. The molecular mechanisms by which DLK proteins regulate cell differentiation and proliferation processes are not fully established yet. In previous reports, we demonstrated that DLK1 interacts with itself and with specific EGF-like repeats of the NOTCH1 extracellular region involved in the binding to NOTCH1 canonical ligands. Moreover, the interaction of DLK1 with NOTCH1 caused an inhibition of basal NOTCH signaling in preadipocytes and mesenchymal multipotent cells. In this work, we demonstrate, for the first time, that DLK2 interacts with itself, with DLK1, and with the same NOTCH1 receptor region as DLK1 does. We demonstrate also that the interaction of DLK2 with NOTCH1 similarly results in an inhibition of NOTCH signaling in preadipocytes and Mouse Embryo fibloblasts. In addition, we demonstrate that a membrane DLK1 variant, lacking the sequence recognized by the protease TACE, also inhibits NOTCH signaling. Furthermore, both DLK1 and DLK2 are able to decrease NOTCH activity also when triggered by specific NOTCH ligands. However, the decrease in NOTCH signaling induced by overexpression of Dlk2 is reversed by the overexpression of Dlk1, and viceversa. We conclude that DLK1 and DLK2 act as inhibitory non-canonical protein ligands for the NOTCH1 receptor that modulate NOTCH signaling.     

The EGF-like proteins DLK1 and DLK2 function as inhibitory non-canonical ligands of NOTCH1 receptor that modulate each other's activities

The protein DLK2, highly homologous to DLK1, belongs to the EGF-like family of membrane proteins, which includes NOTCH receptors and their DSL-ligands. The molecular mechanisms by which DLK proteins regulate cell differentiation and proliferation processes are not fully established yet. In previous reports, we demonstrated that DLK1 interacts with itself and with specific EGF-like repeats of the NOTCH1 extracellular region involved in the binding to NOTCH1 canonical ligands. Moreover, the interaction of DLK1 with NOTCH1 caused an inhibition of basal NOTCH signaling in preadipocytes and mesenchymal multipotent cells. In this work, we demonstrate, for the first time, that DLK2 interacts with itself, with DLK1, and with the same NOTCH1 receptor region as DLK1 does. We demonstrate also that the interaction of DLK2 with NOTCH1 similarly results in an inhibition of NOTCH signaling in preadipocytes and Mouse Embryo fibloblasts. In addition, we demonstrate that a membrane DLK1 variant, lacking the sequence recognized by the protease TACE, also inhibits NOTCH signaling. Furthermore, both DLK1 and DLK2 are able to decrease NOTCH activity also when triggered by specific NOTCH ligands. However, the decrease in NOTCH signaling induced by overexpression of Dlk2 is reversed by the overexpression of Dlk1, and viceversa. We conclude that DLK1 and DLK2 act as inhibitory non-canonical protein ligands for the NOTCH1 receptor that modulate NOTCH signaling.     

Malaria parasites require TLR9 signaling for immune evasion by activating regulatory T cells

Malaria is still a life-threatening infectious disease that continues to produce 2 million deaths annually. Malaria parasites have acquired immune escape mechanisms and prevent the development of sterile immunity. Regulatory T cells (Tregs) have been reported to contribute to immune evasion during malaria in mice and humans, suggesting that activating Tregs is one of the mechanisms by which malaria parasites subvert host immune systems. However, little is known about how these parasites activate Tregs. We herein show that TLR9 signaling to dendritic cells (DCs) is crucial for activation of Tregs. Infection of mice with the rodent malaria parasite Plasmodium yoelii activates Tregs, leading to enhancement of their suppressive function. In vitro activation of Tregs requires the interaction of DCs with parasites in a TLR9-dependent manner. Furthermore, TLR9(-/-) mice are partially resistant to lethal infection, and this is associated with impaired activation of Tregs and subsequent development of effector T cells. Thus, malaria parasites require TLR9 to activate Tregs for immune escape.     

CCL2, survivin and autophagy: new links with implications in human cancer

Recent data strongly support the idea that the orchestrated interaction between cancer and other cells in the tumor microenvironment is a vital component in the neoplastic process. Thus, tumor cells take advantage of the signaling molecules of the immune system to proliferate, survive, and invade other tissues. CCL2 (Chemokine (C-C motif) ligand 2, Monocyte chemoattractant protein-1 (MCP-1) has been demonstrated to play a significant role in prostate cancer neoplasia and invasion, and is highly expressed in the tumor microenvironment. We recently reported that CCL2 elicits a strong survival advantage in prostate cancer PC3 cells through PI3K/Akt-dependent regulation of autophagy via the mammalian target of rapamycin (mTOR) pathway and importantly, survivin upregulation is essential in this survival mechanism. Autophagy protects cells from nutrient depletion stress, but, paradoxically, excessive autophagy will result in cell death. How these life or death decisions are regulated remains unclear. Here we discuss the function of survivin in the control of autophagy and the interaction between CCL2, survivin and autophagy in the complex program of tumor progression.     

The proteins DLK1 and DLK2 modulate NOTCH1-dependent proliferation and oncogenic potential of human SK-MEL-2 melanoma cells

NOTCH receptors regulate cell proliferation and survival in several types of cancer cells. Depending on the cellular context, NOTCH1 can function as an oncogene or as a tumor suppressor gene. DLK1 is also involved in the regulation of cell growth and cancer, but nothing is known about the role of DLK2 in these processes. Recently, the proteins DLK1 and DLK2 have been reported to interact with NOTCH1 and to inhibit NOTCH1 activation and signaling in different cell lines. In this work, we focused on the role of DLK proteins in the control of melanoma cell growth, where NOTCH1 is known to exert an oncogenic effect. We found that human DLK proteins inhibit NOTCH signaling in SK-MEL-2 metastatic melanoma cells. Moreover, the proliferation rate of these cells was dependent upon the level of NOTCH activation and signaling as regulated by DLK proteins. In particular, high levels of NOTCH inhibition resulted in a decrease, whereas lower levels of NOTCH inhibition led to an increase in melanoma cell proliferation rates, both in vitro and in vivo. Finally, our data revealed additive NOTCH-mediated effects of DLK proteins and the γ-secretase inhibitor DAPT on cell proliferation. The data presented in this work suggest that a fine regulation of NOTCH signaling plays an important role in the control of metastatic melanoma cell proliferation. Our results open the way to new research on the role of DLK proteins as potential therapeutic tools for the treatment of human melanoma.     

NOTCH1 regulates the proliferation and migration of bladder cancer cells by cooperating with long non-coding RNA HCG18 and microRNA-34c-5p

In recent years, the NOTCH signaling pathway has been gradually studied in human malignancies. Inactivation of the NOTCH signaling pathway was uncovered to be correlated with the carcinogenesis of bladder cancer (BCa). Nevertheless, the specific molecular mechanism of NOTCH1 (one of the core factors of the NOTCH signaling pathway) is not well elucidated in BCa. This study focused on the mechanism by which NOTCH1 affects the biological behaviors of BCa cells. According to the experimental results of quantitative real-time polymerase chain reaction, NOTCH1 was dysregulated in BCa tissues and cell lines. The prognostic value of NOTCH1 for the patients with BCa was determined using the Kaplan-Meier method. Mechanism investigations revealed that NOTCH1 is a target of miR-34c-5p in BCa. Furthermore, microarray analysis was used to find the dysregulated long noncoding RNAs (lncRNA), which can bind with miR-34c-5p. Mechanism experiments further demonstrated the rationality of the HCG18-miR-34c-5p-NOTCH1 pathway. Functional assays were then applied to validate the inhibitory influences of NOTCH1 on the proliferation and migration of BCa cells. Furthermore, the inhibitory effects of NOTCH1 could be affected by miR-34c-5p or lncRNA HCG18. All findings in this study revealed that NOTCH1 suppresses the BCa progression by cooperating with lncRNA HCG18 and miR-34c-5p.     

The role of the PTEN/AKT Pathway in NOTCH1-induced leukemia

Activating mutations in NOTCH1 are the most prominent genetic abnormality in T-cell acute Lymphoblastic Leukemia (T-ALL) and inhibition of NOTCH1 signaling with γ-secretase inhibitors (GSIs) has been proposed as targeted therapy in this disease. However, most T-ALL cell lines with mutations in NOTCH1 fail to respond to GSI therapy. Using gene expression profiling and mutation analysis we showed that mutational loss of PTEN is a common event in T-ALL and is associated with resistance to NOTCH inhibition. Furthermore, our studies revealed that NOTCH1 induces upregulation of the PI3K-AKT pathway via HES1, which negatively controls the expression of PTEN. This regulatory circuitry is evolutionary conserved from Drosophila to humans as demonstrated by the interaction of overexpression of Delta and Akt in a model of Notch-induced transformation in the fly eye. Loss of PTEN and constitutive activation of AKT in T-ALL induce increased glucose metabolism and bypass the requirement of NOTCH1 signaling to sustain cell growth. Importantly, PTEN-null/GSI resistant T-ALL cells switch their oncogene addiction from NOTCH1 to AKT and are highly sensitive to AKT inhibitors. These results should facilitate the development of molecular therapies targeting NOTCH1 and AKT for the treatment of T-ALL.     

Galactose differentially modulates lunatic and manic fringe effects on Delta1-induced NOTCH signaling

NOTCH signaling induced by Delta1 (DLL1) and Jagged1 (JAG1) NOTCH ligands is modulated by the β3N-acetylglucosaminyl transferase Fringe. LFNG (Lunatic Fringe) and MFNG (Manic Fringe) transfer N-acetylglucosamine (GlcNAc) to O-fucose attached to EGF-like repeats of NOTCH receptors. In co-culture NOTCH signaling assays, LFNG generally enhances DLL1-induced, but inhibits JAG1-induced, NOTCH signaling. In mutant Chinese hamster ovary (CHO) cells that do not add galactose (Gal) to the GlcNAc transferred by Fringe, JAG1-induced NOTCH signaling is not inhibited by LFNG or MFNG. In mouse embryos lacking B4galt1, NOTCH signaling is subtly reduced during somitogenesis. Here we show that DLL1-induced NOTCH signaling in CHO cells was enhanced by LFNG, but this did not occur in either Lec8 or Lec20 CHO mutants lacking Gal on O-fucose glycans. Lec20 mutants corrected with a B4galt1 cDNA became responsive to LFNG. By contrast, MFNG promoted DLL1-induced NOTCH signaling better in the absence of Gal than in its presence. This effect was reversed in Lec8 cells corrected by expression of a UDP-Gal transporter cDNA. The MFNG effect was abolished by a DDD to DDA mutation that inactivates MFNG GlcNAc transferase activity. The binding of soluble NOTCH ligands and NOTCH1/EGF1-36 generally reflected changes in NOTCH signaling caused by LFNG and MFNG. Therefore, the presence of Gal on O-fucose glycans differentially affects DLL1-induced NOTCH signaling modulated by LFNG versus MFNG. Gal enhances the effect of LFNG but inhibits the effect of MFNG on DLL1-induced NOTCH signaling, with functional consequences for regulating the strength of NOTCH signaling.     

The role of regulatory T cells in the pathogenesis of acute kidney injury

The incidence of acute kidney injury (AKI) is on the rise and is associated with high mortality; however, there are currently few effective treatments. Moreover, the relationship between Tregs and other components of the immune microenvironment (IME) in the pathogenesis of AKI remains unclear. We downloaded four publicly accessible AKI datasets, GSE61739, GSE67401, GSE19130, GSE81741, GSE19288 and GSE106993 from the gene expression omnibus (GEO) database. Additionally, we gathered two kidney single-cell sequencing (scRNA-seq) samples from the Department of Organ Transplantation at Zhujiang Hospital of Southern Medical University to investigate chronic kidney transplant rejection (CKTR). Moreover, we also collected three samples of normal kidney tissue from GSE131685. By analysing the differences in immune cells between the AKI and Non-AKI groups, we discovered that the Non-AKI group contained a significantly greater number of Tregs than the AKI group. Additionally, the activation of signalling pathways, such as inflammatory molecules secretion, immune response, glycolytic metabolism, NOTCH, FGF, NF-κB and TLR4, was significantly greater in the AKI group than in the Non-AKI group. Additionally, analysis of single-cell sequencing data revealed that Tregs in patients with chronic kidney rejection and in normal kidney tissue have distinct biology, including immune activation, cytokine production, and activation fractions of signalling pathways such as NOTCH and TLR4. In this study, we found significant differences in the IME between AKI and Non-AKI, including differences in Tregs cells and activation levels of biologically significant signalling pathways. Tregs were associated with lower activity of signalling pathways such as inflammatory response, inflammatory molecule secretion, immune activation, glycolysis.     

Differential Regulation of NOTCH2 and NOTCH3 Contribute to Their Unique Functions in Vascular Smooth Muscle Cells

Notch signaling is a key regulator of vascular smooth muscle cell (VSMC) phenotypes, including differentiation, proliferation, and cell survival. However, the exact contribution of the individual Notch receptors has not been thoroughly delineated. In this study, we identify unique roles for NOTCH2 and NOTCH3 in regulating proliferation and cell survival in cultured VSMCs. Our results indicate that NOTCH2 inhibits PDGF-B-dependent proliferation and its expression is decreased by PDGF-B. In contrast, NOTCH3 promotes proliferation and receptor expression is increased by PDGF-B. Additionally, data show that NOTCH3, but not NOTCH2 protects VSMCs from apoptosis and apoptosis mediators degrade NOTCH3 protein. We identified three pro-survival genes specifically regulated by NOTCH3 in cultured VSMCs and in mouse aortas. This regulation is mediated through MAP kinase signaling, which we demonstrate can be activated by NOTCH3, but not NOTCH2. Overall, this study highlights discrete roles for NOTCH2 and NOTCH3 in VSMCs and connects these roles to specific upstream regulators that control their expression.     

CCL2, survivin and autophagy: New links with implications in human cancer

Recent data strongly support the idea that the orchestrated interaction between cancer and other cells in the tumor microenvironment is a vital component in the neoplastic process. Thus, tumor cells take advantage of the signaling molecules of the immune system to proliferate, survive and invade other tissues. CCL2 (Chemokine (C-C motif) ligand 2, Monocyte chemoattractant protein-1 (MCP-1)) has been demonstrated to play a significant role in prostate cancer neoplasia and invasion, and is highly expressed in the tumor microenvironment. We recently reported that CCL2 elicits a strong survival advantage in prostate cancer PC3 cells through PI3K/Akt-dependent regulation of autophagy via the mammalian target of rapamycin (mTOR) pathway and importantly, survivin up-regulation is essential to this survival mechanism. Autophagy protects cells from nutrient depletion stress, but, paradoxically, excessive autophagy will result in cell death. How these life or death decisions are regulated remains unclear. Here we discuss the function of survivin in the control of autophagy and the interaction between CCL2, survivin and autophagy in the complex program of tumor progression.

Addendum to: Roca H, Varsos Z, Pienta KJ. CCL2 protects prostate cancer PC3 cells from autophagic death via PI3K/AKT-dependent survivin up-regulation. J Biol Chem 2008; In press.     

NOTCH1 Gain of Function in Germ Cells Causes Failure of Spermatogenesis in Male Mice

NOTCH1 is a member of the NOTCH receptor family, a group of single-pass trans-membrane receptors. NOTCH signaling is highly conserved in evolution and mediates communication between adjacent cells. NOTCH receptors have been implicated in cell fate determination, as well as maintenance and differentiation of stem cells. In the mammalian testis expression of NOTCH1 in somatic and germ cells has been demonstrated, however its role in spermatogenesis was not clear. To study the significance of NOTCH1 in germ cells, we applied a cre/loxP approach in mice to induce NOTCH1 gain- or loss-of function specifically in male germ cells. Using a Stra8-icre transgene we produced mice with conditional activation of the NOTCH1 intracellular domain (NICD) in germ cells. Spermatogenesis in these mutants was progressively affected with age, resulting in decreased testis weight and sperm count. Analysis of downstream target genes of NOTCH1 signaling showed an increased expression of Hes5, with a reduction of the spermatogonial differentiation marker, Neurog3 expression in the mutant testis. Apoptosis was significantly increased in mouse germ cells with the corresponding elevation of pro-apoptotic Trp53 and Trp63 genes'' expression. We also showed that the conditional germ cell-specific ablation of Notch1 had no effect on spermatogenesis or male fertility. Our data suggest the importance of NOTCH signaling regulation in male germ cells for their survival and differentiation.     

Autophagy in regulation of Toll-like receptor signaling

Toll-like receptors (TLRs) serve as the major innate immune sensors for detection of specific molecular patterns on various pathogens. TLRs activate signaling events mainly by utilizing ubiquitin-dependent mechanisms. Recent research advances have provided evidence that TLR signaling is linked to induction of autophagy. Autophagy is currently known to affect both of the immune defense and suppression of inflammatory responses. In TLR-associated immune responses, autophagic lysis of intracellular microbes (called xenophagy) contributes to the former mechanism, while the latter seems to be mediated by the control of the mitochondrial integrity or selective autophagic clearance of aggregated signaling proteins (called aggrephagy). Several autophagy-related ubiquitin-binding proteins, such as SQSTM1/p62 and NDP52, mediate xenophagy and aggrephagy. In this review, we summarize the expanded knowledge regarding TLR signaling and autophagy signaling. After that, we will focus on autophagy-associated signaling downstream of TLRs and the effect of autophagy on TLR signaling, thus highlighting the signaling crosstalk between the TLR-associated innate immune responses and the regulation of innate immunity by xenophagy and aggrephagy.     

Peritumoral monocytes induce cancer cell autophagy to facilitate the progression of human hepatocellular carcinoma

Macroautophagy/autophagy is an important catabolic process mediating cellular homeostasis and plays critical roles in cancer development. Whereas autophagy has been widely studied in various pathological models, little is known about the distribution, clinical significance and regulatory mechanism of this process in human hepatocellular carcinoma (HCC). In the present study, we found that tumor tissues exhibited significantly increased levels of autophagy compared with non-tumor tissues, and cancer cells with higher levels of autophagy were predominantly enriched in the invading edge regions of human HCC. Increased MAP1LC3B/LC3B expression in the invading edge regions was significantly correlated with a higher density of closely located monocytes, and TNF and IL1B derived from tumor-activated monocytes synergistically induced cancer cell autophagy in the invading edge regions of HCC. Monocyte-elicited autophagy induced the epithelial-mesenchymal transition (EMT) of cancer cells and promoted tumor metastasis by activating the NFKB-SNAI1 signaling pathway. Moreover, the increase of LC3B⁺ cancer cells in the invading edge areas was associated with high mortality and reduced survival of patients with HCC. These findings indicated that cancer cell autophagy is regulated by a collaborative interaction between tumor and immune cell components in distinct HCC microenvironments, thus allowing the inflammatory monocytes to be rerouted in a tumor-promoting direction.     

NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma

Recent studies have shown that Notch signaling is involved in many types of cancers, including oral squamous cell carcinomas (OSCCs). However, the role of Notch signaling in the tumor microenvironment is not yet fully understood. In this study, we investigated the roles of NOTCH3 signaling in cancer associated fibroblasts (CAFs) in OSCCs. Immunohistochemical study of 93 human tongue OSCC cases indicated that about one third of OSCCs showed NOTCH3 expression in CAFs, and that this expression significantly correlated with tumor-size. In vitro study showed that OSCC cell lines, especially HO1-N-1 cells stimulated NOTCH3 expression in normal human dermal fibroblasts (NHDFs) through direct cell-to-cell contact. Immunohistochemical and morphometric analysis using human OSCC samples demonstrated that NOTCH3 expression in CAFs significantly correlated with micro-vessel density in cancer stroma. In vitro angiogenesis assays involving co-culture of NHDFs with HO1-N-1 and human umbilical endothelial cells (HUVECs), and NOTCH3 knockdown in NHDFs using siRNA, demonstrated that HO1-N-1 cells significantly promoted tube formation dependent on NOTCH3-expression in NHDFs. Moreover, NOTCH3 expression in CAFs was related to poor prognosis of the OSCC patients. This work provides a new insight into the role of Notch signaling in CAFs associated with tumor angiogenesis and the possibility of NOTCH3-targeted molecular therapy in OSCCs.     

How to teach an old dog new tricks: Autophagy-independent action of chloroquine on the tumor vasculature

Chloroquine (CQ) is exploited in clinical trials as an autophagy blocker to potentiate anticancer therapy, but it is unknown if it solely acts by inhibiting cancer cell-autonomous autophagy. Our recent study shows that besides blocking cancer cell growth, CQ also affects endothelial cells (ECs) and promotes tumor vessel normalization. This vessel normalizing effect of CQ reduces tumor hypoxia, cancer cell intravasation, and metastasis, while improving the delivery and response to chemotherapy. By compromising autophagy in melanoma cells or using mice with a conditional knockout of ATG5 in ECs, we found that the favorable effects of CQ on the tumor vasculature do not rely on autophagy. CQ-induced vessel normalization relies mainly on altered endolysosomal trafficking and sustained NOTCH1 signaling in ECs. Remarkably these CQ-mediated effects are abrogated when tumors are grown in mice harboring EC-specific deletion of NOTCH1. The autophagy-independent vessel normalization by CQ leading to improved delivery and tumor response to chemotherapy further advocates its clinical use in combination with anticancer treatments.     

Regulation of macroautophagy by mTOR and Beclin 1 complexes

Macroautophagy or autophagy is a vacuolar degradative pathway terminating in the lysosomal compartment after forming a cytoplasmic vacuole or autophagosome that engulfs macromolecules and organelles. The original discovery that ATG (AuTophaGy related) genes in yeast are involved in the formation of autophagosomes has greatly increased our knowledge of the molecular basis of autophagy, and its role in cell function that extends far beyond non-selective degradation. The regulation of autophagy by signaling pathways overlaps the control of cell growth, proliferation, cell survival and death. The evolutionarily conserved TOR (Target of Rapamycin) kinase complex 1 plays an important role upstream of the Atg1 complex in the control of autophagy by growth factors, nutrients, calcium signaling and in response to stress situations, including hypoxia, oxidative stress and low energy. The Beclin 1 (Atg6) complex, which is involved in the initial step of autophagosome formation, is directly targeted by signaling pathways. Taken together, these data suggest that multiple signaling checkpoints are involved in regulating autophagosome formation.