Thomas Naves

Autophagy in human keratinocytes: an early step of the differentiation?

Abstract:  Studies have established that autophagy constitutes an efficient process to recycle cellular components and certain proteins. The phenomenon was demonstrated primarily in response to nutrient starvation, and there are increasing evidences that it is implied in differentiation. Keratinocyte differentiation was going along an activation of lysosomal enzymes and organelle clearance, and terminal steps are sometimes described as a specialized form of cell death leading to corneocytes. We examined whether initiation of the process in human keratinocyte HaCaT involves autophagy. The KSFM™ culture medium was substituted by M199, which contains a low glucose concentration but a high calcium level (known to induce differentiation). Metabolic stress reduced enhanced cell number in G1 phase, without apoptotic features (ΔΨmt and membrane integrity are unchanged). Morphological changes were associated with a lower integrin ß1 expression and modifications of protein levels involved in keratinocyte differentiation (involucrin, keratin K10 and ΔNp63α). Whereas autophagic signalling was supported by SIRT1 and pAMPK (T172) increase according to time kinetic, which led to the disappearance of mTOR phosphorylated on S2448 residue. The significant Bcl‐XL level reduction with stress promoted autophagy, by the release of Beclin‐1, whereas ATG5‐ATG12 and LC3‐II that are involved in autophagosome formation were enhanced significantly. Then, the level of lysosomal protein cathepsin B rose to execute autophagy. Kinetic studies established that autophagy would constitute an early signalling process required for keratinocyte commitment in differentiation pathway.

Sortilin mediates the release and transfer of exosomes in concert with two tyrosine kinase receptors

ABSTRACT The transfer of exosomes containing both genetic and protein materials is necessary for the control of the cancer cell microenvironment to promote tumor angiogenesis. The nature and function of proteins found in the exosomal cargo, and the mechanism of their action in membrane transport and related signaling events are not clearly understood. In this study, we demonstrate, in human lung cancer A549 cells, that the exosome release mechanism is closely linked to the multifaceted receptor sortilin (also called neurotensin receptor 3). Sortilin is already known to be important for cancer cell function. Here, we report for the first time its role in the assembly of a tyrosine kinase complex and subsequent exosome release. This new complex (termed the TES complex) is found in exosomes and results in the linkage of the two tyrosine kinase receptors TrkB (also known as NTRK2) and EGFR with sortilin. Using in vitro models, we demonstrate that this sortilin-containing complex exhibits a control on endothelial cells and angiogenesis activation through exosome transfer.

Autophagy takes place in mutated p53 neuroblastoma cells in response to hypoxia mimetic CoCl2

Solid tumors like neuroblastoma exhibit hypoxic areas, which can lead both to cell death or aggressiveness increase. Hypoxia is a known stress able to induce stabilization of p53, implicated in cell fate regulation. Recently, p53 appeared to be involved in autophagy in an opposite manner, depending on its location: when nuclear, it enhanced transcription of pro-autophagic genes whereas when cytoplasmic, it inhibited the autophagic process. Today, we used cobalt chloride, a hypoxia mimetic that inhibits proteasomal HIF-1 degradation and generates reactive oxygen species (ROS). We focused on CoCl2-induced cell death in a DNA-binding mutated p53 neuroblastoma cell line (SKNBE(2c)). An autophagic signaling was evidenced by an increase of Beclin-1, ATG 5-12, and LC3-II expression whereas the p53(mut) presence decreased with CoCl2 time exposure. Activation of the pathway seemed to protect cells from ROS production and, at least in part, from death. The autophagic inhibitors activated the apoptotic signaling and the death was enhanced. To delineate the eventual implication of the p53(mut) in the autophagic process in response to hypoxia, we monitored signaling in p53(WT)SHSY5Y cells, after either shRNA-p53 down-regulation or transcriptional activity inhibition by pifithrin alpha. We did not detect autophagy neither with p53(wt) nor when p53 was lacking whereas such a response was effective with a mutated or inactivated p53. To conclude, mutated p53 in neuroblastoma cells could be linked with the switch between apoptotic response and cell death by autophagy in response to hypoxic mimetic stress.

The cell death response to the ROS inducer, cobalt chloride, in neuroblastoma cell lines according to p53 status.

Cobalt chloride (CoCl2), a hypoxia-mimetic agent, induces reactive oxygen species (ROS) generation, leading to cell death. Divergent data have been reported concerning p53 implication in this apoptotic mechanism. In this study, we studied cobalt-induced cell death in neuroblastoma cell lines carrying wild-type (WT) p53 ( SHSY5Y) and a mutated DNA-binding domain p53 [SKNBE(2c)]. CoCl2 induced an upregulation of p53, p21 and PUMA expression in WT cells but not in SKNBE(2c). In SHSY5Y cells, p53 serine-15 phosphorylation appeared early (6 h) in the mitochondria, and also in the nucleus after 12 h. In contrast, in SKNBE(2c) cells, the slight nuclear signal disappeared with CoCl2 treatment. In SHSY5Y cells, a mitochondrial pathway dependent on caspases [collapse of mitochondrial transmembrane potential (∆Ψmt), caspase 3 and 9 activation], was activated in a time-dependent manner. SKNBE(2c) cells exhibited a delay in the cell death executive phase linked to a caspase-independent pathway, involving apoptosis inducing factor nuclear translocation, but also an autophagic process attested by LC3-II expression and cathepsin-B activation. The downregulation of p53 in SHSY5Y cells by siRNA induced a cell death pathway related to the one observed in SKNBE(2c) cells. Finally, CoCl2 induced time-dependent canonical p53 mitochondrial apoptosis in the WT p53 cell line, and caspase-independent cell death in cells with a mutated or KO p53.

The implications of sortilin/vps10p domain receptors in neurological and human diseases.

The neurotensin receptor-3 also known as sortilin is part of the new receptor family of vacuolar protein sorting 10 protein domain. Growing evidence show that the vacuolar protein sorting 10 protein domain family is implicated as a genetic risk factor for neurodegenerative diseases such as Alzheimers disease, frontotemporal lobar degeneration, and Parkinsons disease, in addition to links associated with type 2 diabetes mellitus, lysosomal disorders, cardiovascular disease and atherosclerosis. In fact, sortilin expression is elevated in many human cell lines controlling the trafficking and release of neurotrophins. Hence, not surprisingly the imbalance of neurotrophin signaling is implicated in several human diseases. The fine regulation of the growth factor, brain derived nerve factor by sortilin mediates both neuronal and tumor cell survival, whereas in Alzheimers disease sortilin mediated beta secretase-1 trafficking increases the cleavage of the beta-amyloid precursor protein. Perturbation of the autocrine/paracrine loop of neurotrophins in combination with the cell surface interaction of sortilin with neurotensin receptor 1 or 2 or tyrosine kinase receptor A or B are dramatically upregulated in both neurodegenerative diseases and cancer. In cardiovascular diseases, the circulatory low-density lipoprotein is closely correlated with sortilin expression in hepatocytes. Herein, this review discusses the multifaceted role played by sortilin and its interacting partners in human disease which could be interesting novel target(s) in drug discovery. Nevertheless, completely challenging the function of sortilin could prove unfavorable given the important universal role of sortilin plays in the body. Hence, metabolism disorders could be relieved with specific targeted therapeutic challenge of sortilin function.

p75 neurotrophin receptor and pro-BDNF promote cell survival and migration in clear cell renal cell carcinoma

p75NTR, a member of TNF receptor family, is the low affinity receptor common to several mature neurotrophins and the high affinity receptor for pro-neurotrophins. Brain-Derived Neurotrophic Factor (BDNF), a member of neurotrophin family has been described to play an important role in development and progression of several cancers, through its binding to a high affinity tyrosine kinase receptor B (TrkB) and/or p75NTR. However, the functions of these two receptors in renal cell carcinoma (RCC) have never been investigated. An overexpression of p75NTR, pro-BDNF, and to a lesser extent for TrkB and sortilin, was detected by immunohistochemistry in a cohort of 83 clear cell RCC tumors. p75NTR, mainly expressed in tumor tissues, was significantly associated with higher Fuhrman grade in multivariate analysis. In two derived-RCC lines, 786-O and ACHN cells, we demonstrated that pro-BDNF induced cell survival and migration, through p75NTR as provided by p75NTR RNA silencing or blocking anti-p75NTR antibody. This mechanism is independent of TrkB activation as demonstrated by k252a, a tyrosine kinase inhibitor for Trk neurotrophin receptors. Taken together, these data highlight for the first time an important role for p75NTR in renal cancer and indicate a putative novel target therapy in RCC.

The Ins and Outs of Nanoparticle Technology in Neurodegenerative Diseases and Cancer.

As we enter the twenty-first century, several therapies based on using nanoparticles (NPs) ranging in size 1 - 1000 nm have been successfully brought to the clinic to treat cancer, pain and infectious diseases. These therapies bring together the ability of NPs to target the delivery of drugs more precisely, to improve solubility, to prevent degradation, to improve their therapeutic index and to reduce the immune response. NPs come in all shapes and sizes, designed specifically for biomedical applications such as solid lipid polymers, liposomes, dendrimers, nanogels, and quantum dots. These NPs offer many attractive characteristics such as biological stability and biocompatibility, thus incorporating different biological or drug molecules. Among the major therapeutic challenges from neurological diseases through to cancer is the development of nanomaterials that are able to be effective against the disease. In the case of neurodegeneration, one of the most difficult areas to penetrate for drug discovery in the body is the central nervous system, protected by the blood-brain-barrier. Whilst in the case of cancer, the biggest problem is how to specifically target a tumor with sufficient drug without causing side effects or inducing resistance. A new generation of intelligent NPs are emerging for the treatment of human disease such as neurological disorders and cancer. The use of natural alternative therapy is an encouraging idea in drug discovery. To this end as we gain more knowledge into the biological function of exosomes, this will allow us to harness their potential as natural NPs in future therapeutics.

A new role under sortilin's belt in cancer

ABSTRACT The neurotensin receptor-3 also known as sortilin was the first member of the small family of vacuolar protein sorting 10 protein domain (Vps10p) discovered two decades ago in the human brain. The expression of sortilin is not confined to the nervous system but sortilin is ubiquitously expressed in many tissues. Sortilin has multiple roles in the cell as a receptor or a co-receptor, in protein transport of many interacting partners to the plasma membrane, to the endocytic pathway and to the lysosomes for protein degradation. Sortilin could be considered as the cells own shuttle system. In many human diseases including neurological diseases and cancer, sortilin expression has been shown to be deregulated. In addition, some studies have highlighted that the extracellular domain of sortilin is shedded into the culture media by an unknown mechanism. Sortilin can be released in exosomes and appears to control some mechanisms of exosome biogenesis. In lung cancer cells, sortilin can associate with two receptor tyrosine kinase receptors called the TES complex found in exosomes. Exosomes carrying the TES complex can convey a microenvironment control through the activation of ErbB signaling pathways and the release of angiogenic factors. Deregulation of sortilin function is now emerging to be implicated in four major human diseases- cardiovascular disease, Type 2 diabetes mellitus, Alzheimer disease and cancer.

Sortilin limits EGFR signaling by promoting its internalization in lung cancer

Tyrosine kinase receptors such as the epidermal growth factor receptor (EGFR) transduce information from the microenvironment into the cell and activate homeostatic signaling pathways. Internalization and degradation of EGFR after ligand binding limits the intensity of proliferative signaling, thereby helping to maintain cell integrity. In cancer cells, deregulation of EGFR trafficking has a variety of effects on tumor progression. Here we report that sortilin is a key regulator of EGFR internalization. Loss of sortilin in tumor cells promoted cell proliferation by sustaining EGFR signaling at the cell surface, ultimately accelerating tumor growth. In lung cancer patients, sortilin expression decreased with increased pathologic grade, and expression of sortilin was strongly correlated with survival, especially in patients with high EGFR expression. Sortilin is therefore a regulator of EGFR intracellular trafficking that promotes receptor internalization and limits signaling, which in turn impacts tumor growth.Disruptions in epidermal growth factor receptor (EGFR) trafficking has been linked to tumor progression. Here the authors show that sortilin limits cell proliferation and tumor growth by promoting EGFR internalization.

Neurotensin receptor type 2 protects B-cell chronic lymphocytic leukemia cells from apoptosis

B-cell chronic lymphocytic leukemia (B-CLL) cells are resistant to apoptosis, and consequently accumulate to the detriment of normal B cells and patient immunity. Because current therapies fail to eradicate these apoptosis-resistant cells, it is essential to identify alternative survival pathways as novel targets for anticancer therapies. Overexpression of cell-surface G protein-coupled receptors drives cell transformation, and thus plays a critical role in malignancies. In this study, we identified neurotensin receptor 2 (NTSR2) as an essential driver of apoptosis resistance in B-CLL. NTSR2 was highly expressed in B-CLL cells, whereas expression of its natural ligand, neurotensin (NTS), was minimal in both B-CLL cells and patient plasma. Surprisingly, NTSR2 remained in a constitutively active phosphorylated state, caused not by a mutation-induced gain-of-function but rather by an interaction with the oncogenic tyrosine kinase receptor TrkB. Functional and biochemical characterization revealed that the NTSR2–TrkB interaction acts as a conditional oncogenic driver requiring the TrkB ligand brain-derived neurotrophic factor (BDNF), which unlike NTS is highly expressed in B-CLL cells. Together, NTSR2, TrkB and BDNF induce autocrine and/or paracrine survival pathways that are independent of mutation status and indolent or progressive disease course. The NTSR2–TrkB interaction activates survival signaling pathways, including the Src and AKT kinase pathways, as well as expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL. When NTSR2 was downregulated, TrkB failed to protect B-CLL cells from a drastic decrease in viability via typical apoptotic cell death, reflected by DNA fragmentation and Annexin V presentation. Together, our findings demonstrate that the NTSR2–TrkB interaction plays a crucial role in B-CLL cell survival, suggesting that inhibition of NTSR2 represents a promising targeted strategy for treating B-CLL malignancy.