Bivalent EGFR-Targeting DARPin-MMAE Conjugates.

Epidermal growth factor receptor (EGFR) is a validated tumor marker overexpressed in various cancers such as squamous cell carcinoma (SSC) of the head and neck and gliomas. We constructed protein-drug conjugates based on the anti-EGFR Designed Ankyrin Repeat Protein (DARPin) E01, and compared the bivalent DARPin dimer (DD1) and a DARPin-Fc (DFc) to the monomeric DARPin (DM) and the antibody derived scFv425-Fc (scFvFc) in cell culture and a mouse model. The modular conjugation system, which was successfully applied for the preparation of protein-drug and -dye conjugates, uses bio-orthogonal protein-aldehyde generation by the formylglycine-generating enzyme (FGE). The generated carbonyl moiety is addressed by a bifunctional linker with a pyrazolone for a tandem Knoevenagel reaction and an azide for strain-promoted azide-alkyne cycloaddition (SPAAC). The latter reaction with a PEGylated linker containing a dibenzocyclooctyne (DBCO) for SPAAC and monomethyl auristatin E (MMAE) as the toxin provided the stable conjugates DD1-MMAE (drug-antibody ratio, DAR = 2.0) and DFc-MMAE (DAR = 4.0) with sub-nanomolar cytotoxicity against the human squamous carcinoma derived A431 cells. In vivo imaging of Alexa Fluor 647-dye conjugates in A431-xenografted mice bearing subcutaneous tumors as the SCC model revealed unspecific binding of bivalent DARPins to the ubiquitously expressed EGFR. Tumor-targeting was verified 6 h post-injection solely for DD1 and scFvFc. The total of four administrations of 6.5 mg/kg DD1-MMAE or DFc-MMAE twice weekly did not cause any sequela in mice. MMAE conjugates showed no significant anti-tumor efficacy in vivo, but a trend towards increased necrotic areas (p = 0.2213) was observed for the DD1-MMAE (n = 5).

Prostate-specific membrane antigen expression in the vasculature of primary lung carcinomas associates with faster metastatic dissemination to the brain.

Glioblastomas and brain metastases (BM) of solid tumours are the most common central nervous system neoplasms associated with very unfavourable prognosis. In this study, we report the association of prostate-specific membrane antigen (PSMA) with various clinical parameters in a large cohort of primary and secondary brain tumours. A tissue microarray containing 371 cases of ascending grades of gliomas pertaining to astrocytic origin and samples of 52 cases of primary lung carcinomas with matching BM with follow-up time accounting to 10.4 years was evaluated for PSMA expression using immunohistochemistry. In addition, PSMA expression was studied in BM arising from melanomas and breast carcinomas. Neovascular expression of PSMA was evident alongside with high expression in the proliferating microvasculature of glioblastomas when compared to the tumour cell expression. This result correlated with the results obtained from the in silico (cancer genome databases) analyses. In gliomas, only the vascular expression of PSMA associated with poor overall survival but not the tumour cell expression. In the matched primary lung cancers and their BM (n = 52), vascular PSMA expression in primary tumours associated with significantly accelerated metastatic dissemination to the brain with a tendency towards poor overall survival. Taken together, we report that the vascular expression of PSMA in the primary and secondary brain tumours globally associates with the malignant progression and poor outcome of the patients.

Seek & Destroy, use of targeting peptides for cancer detection and drug delivery.

Accounting for 16 million new cases and 9 million deaths annually, cancer leaves a great number of patients helpless. It is a complex disease and still a major challenge for the scientific and medical communities. The efficacy of conventional chemotherapies is often poor and patients suffer from off-target effects. Each neoplasm exhibits molecular signatures - sometimes in a patient specific manner - that may completely differ from the organ of origin, may be expressed in markedly higher amounts and/or in different location compared to the normal tissue. Although adding layers of complexity in the understanding of cancer biology, this cancer-specific signature provides an opportunity to develop targeting agents for early detection, diagnosis, and therapeutics. Chimeric antibodies, recombinant proteins or synthetic polypeptides have emerged as excellent candidates for specific homing to peripheral and central nervous system cancers. Specifically, peptide ligands benefit from their small size, easy and affordable production, high specificity, and remarkable flexibility regarding their sequence and conjugation possibilities. Coupled to imaging agents, chemotherapies and/or nanocarriers they have shown to increase the on-site delivery, thus allowing better tumor mass contouring in imaging and increased efficacy of the chemotherapies associated with reduced adverse effects. Therefore, some of the peptides alone or in combination have been tested in clinical trials to treat patients. Peptides have been well-tolerated and shown absence of toxicity. This review aims to offer a view on tumor targeting peptides that are either derived from natural peptide ligands or identified using phage display screening. We also include examples of peptides targeting the high-grade malignant tumors of the central nervous system as an example of the complex therapeutic management due to the tumor's location. Peptide vaccines are outside of the scope of this review.

Synthesis, biological evaluation, and in vivo imaging of the first camptothecin-fluorescein conjugate.

The first synthesis and photophysical properties of a fluorecently labeled camptothecin derivative, namely, camptothecin-FI (CPT-FI), an antitumoral agent that targets topoisomerase I, are reported. The preparation of this fluorescent conjugate is based on a highly convergent and flexible approach which enables the rapid chemical modification of the AB ring system of this fragile pentacyclic alkaloid, aimed at introducing an anchoring point to graft the fluorophore. The selection of a fluorescein analogue as the reporter group has enabled us to get the first green-emitting CPT conjugate exhibiting valuable spectral properties and retaining biological properties of native CPT. Indeed, in biological models, i.e., glioma cell lines U87 and/or T98, the kinetics of cell endocytosis, as well as the efficacy of CPT-FI were compared to those of CPT. CPT-FI fluorescence was measured in the cytosolic compartment of T98 glioma cells from 5 min treatment and remained detectable until 48 h. As CPT, CPT-FI drastically inhibited glioma growth and cell cycle but exhibited a reduced affinity as compared to the native CPT. In vivo and ex vivo imaging studies of CPT-FI intratumoraly injected into a model of NIH-3T3 murine tumor xenografts in nude mice, showed accumulation around the injected site area, which is very promising to target tumors and follow biodistribution in vivo.

Synthesis and biological evaluation of a novel (99m)Tc labeled 2-nitroimidazole derivative as a potential agent for imaging tumor hypoxia.

Tumor hypoxia plays a major role in reducing the efficacy of therapeutic modalities like chemotherapy and radiation therapy in combating cancer. In order to target hypoxic tissues, a tripeptide ligand having a 2-nitroimidazole moiety, as a bioreductive species, was synthesized. The latter was radiolabeled with (99m)Tc for imaging hypoxic regions of tumors and was characterized by means of its rhenium analogue. The biodistribution and scintigraphic image of the corresponding (99m)Tc-complex showed accumulation in tumor and these results suggest that it could be a marker for imaging tumor hypoxia.

Circumventing Drug Treatment? Intrinsic Lethal Effects of Polyethyleneimine (PEI)-Functionalized Nanoparticles on Glioblastoma Cells Cultured in Stem Cell Conditions.

Glioblastoma (GB) is the most frequent malignant tumor originating from the central nervous system. Despite breakthroughs in treatment modalities for other cancer types, GB remains largely irremediable due to the high degree of intratumoral heterogeneity, infiltrative growth, and intrinsic resistance towards multiple treatments. A sub-population of GB cells, glioblastoma stem cells (GSCs), act as a reservoir of cancer-initiating cells and consequently, constitute a significant challenge for successful therapy. In this study, we discovered that PEI surface-functionalized mesoporous silica nanoparticles (PEI-MSNs), without any anti-cancer drug, very potently kill multiple GSC lines cultured in stem cell conditions. Very importantly, PEI-MSNs did not affect the survival of established GB cells, nor other types of cancer cells cultured in serum-containing medium, even at 25 times higher doses. PEI-MSNs did not induce any signs of apoptosis or autophagy. Instead, as a potential explanation for their lethality under stem cell culture conditions, we demonstrate that the internalized PEI-MSNs accumulated inside lysosomes, subsequently causing a rupture of the lysosomal membranes. We also demonstrate blood-brain-barrier (BBB) permeability of the PEI-MSNs in vitro and in vivo. Taking together the recent indications for the vulnerability of GSCs for lysosomal targeting and the lethality of the PEI-MSNs on GSCs cultured under stem cell culture conditions, the results enforce in vivo testing of the therapeutic impact of PEI-functionalized nanoparticles in faithful preclinical GB models.

CD109-GP130 interaction drives glioblastoma stem cell plasticity and chemoresistance through STAT3 activity.

Glioma stem cells (GSCs) drive propagation and therapeutic resistance of glioblastomas, the most aggressive diffuse brain tumors. However, the molecular mechanisms that maintain the stemness and promote therapy resistance remain poorly understood. Here we report CD109/STAT3 axis as crucial for the maintenance of stemness and tumorigenicity of GSCs and as a mediator of chemoresistance. Mechanistically, CD109 physically interacts with glycoprotein 130 to promote activation of the IL-6/STAT3 pathway in GSCs. Genetic depletion of CD109 abolished the stemness and self-renewal of GSCs and impaired tumorigenicity. Loss of stemness was accompanied with a phenotypic shift of GSCs to more differentiated astrocytic-like cells. Importantly, genetic or pharmacologic targeting of CD109/STAT3 axis sensitized the GSCs to chemotherapy, suggesting that targeting CD109/STAT3 axis has potential to overcome therapy resistance in glioblastoma.

Redox responsive Pluronic micelle mediated delivery of functional siRNA: a modular nano-assembly for targeted delivery.

There is an unmet need to develop strategies that allow site-specific delivery of short interfering RNA (siRNA) without any associated toxicity. To address this challenge, we have developed a novel siRNA delivery platform using chemically modified pluronic F108 as an amphiphilic polymer with a releasable bioactive disulfide functionality. The micelles exhibited thermoresponsive properties and showed a hydrodynamic size of ∼291 nm in DLS and ∼200-250 nm in SEM at 37 °C. The grafting of free disulfide pyridyl groups enhanced the transfection efficiency and was successfully demonstrated in human colon carcinoma (HCT116; 88%) and glioma cell lines (U87; 90%), non-cancerous human dermal fibroblast (HDF; 90%) cells as well as in mouse embryonic stem (mES; 54%) cells. To demonstrate the versatility of our modular nanocarrier design, we conjugated the MDGI receptor targeting COOP peptide on the particle surface that allowed the targeted delivery of the cargo molecules to human patent-derived primary BT-13 gliospheres. Transfection experiments with this design resulted in ∼65% silencing of STAT3 mRNA in BT-13 gliospheres, while only ∼20% of gene silencing was observed in the absence of the peptide. We believe that our delivery method solves current problems related to the targeted delivery of RNAi drugs for potential in vivo applications.

Peptidotriazolamers Inhibit Aß(1-42) Oligomerization and Cross a Blood-Brain-Barrier Model.

In peptidotriazolamers every second peptide bond is replaced by a 1H-1,2,3-triazole. Such foldamers are expected to bridge the gap in molecular weight between small-molecule drugs and protein-based drugs. Amyloid ß (Aß) aggregates play an important role in Alzheimer's disease. We studied the impact of amide bond replacements by 1,4-disubstituted 1H-1,2,3-triazoles on the inhibitory activity of the aggregation "hot spots" K16 LVFF20 and G39 VVIA42 in Aß(1-42). We found that peptidotriazolamers act as modulators of the Aß(1-42) oligomerization. Some peptidotriazolamers are able to interfere with the formation of toxic early Aß oligomers, depending on the position of the triazoles, which is also supported by computational studies. Preliminary in vitro results demonstrate that a highly active peptidotriazolamer is also able to cross the blood-brain-barrier.

Targeting the Urotensin II/UT G Protein-Coupled Receptor to Counteract Angiogenesis and Mesenchymal Hypoxia/Necrosis in Glioblastoma.

Glioblastomas (GBMs) are the most common primary brain tumors characterized by strong invasiveness and angiogenesis. GBM cells and microenvironment secrete angiogenic factors and also express chemoattractant G protein-coupled receptors (GPCRs) to their advantage. We investigated the role of the vasoactive peptide urotensin II (UII) and its receptor UT on GBM angiogenesis and tested potential ligand/therapeutic options based on this system. On glioma patient samples, the expression of UII and UT increased with the grade with marked expression in the vascular and peri-necrotic mesenchymal hypoxic areas being correlated with vascular density. In vitro human UII stimulated human endothelial HUV-EC-C and hCMEC/D3 cell motility and tubulogenesis. In mouse-transplanted Matrigel sponges, mouse (mUII) and human UII markedly stimulated invasion by macrophages, endothelial, and smooth muscle cells. In U87 GBM xenografts expressing UII and UT in the glial and vascular compartments, UII accelerated tumor development, favored hypoxia and necrosis associated with increased proliferation (Ki67), and induced metalloproteinase (MMP)-2 and -9 expression in Nude mice. UII also promoted a "tortuous" vascular collagen-IV expressing network and integrin expression mainly in the vascular compartment. GBM angiogenesis and integrin αvß3 were confirmed by in vivo 99mTc-RGD tracer imaging and tumoral capture in the non-necrotic area of U87 xenografts in Nude mice. Peptide analogs of UII and UT antagonist were also tested as potential tumor repressor. Urotensin II-related peptide URP inhibited angiogenesis in vitro and failed to attract vascular and inflammatory components in Matrigel in vivo. Interestingly, the UT antagonist/biased ligand urantide and the non-peptide UT antagonist palosuran prevented UII-induced tubulogenesis in vitro and significantly delayed tumor growth in vivo. Urantide drastically prevented endogenous and UII-induced GBM angiogenesis, MMP, and integrin activations, associated with GBM tumoral growth. These findings show that UII induces GBM aggressiveness with necrosis and angiogenesis through integrin activation, a mesenchymal behavior that can be targeted by UT biased ligands/antagonists.

Prolyl 4-hydroxylase subunit alpha 1 (P4HA1) is a biomarker of poor prognosis in primary melanomas, and its depletion inhibits melanoma cell invasion and disrupts tumor blood vessel walls.

Melanoma is an unpredictable, highly metastatic malignancy, and treatment of advanced melanoma remains challenging. Novel molecular markers based on the alterations in gene expression and the molecular pathways activated or deactivated during melanoma progression are needed for predicting the course of the disease already in primary tumors and for providing new targets for therapy. Here, we sought to identify genes whose expression in primary melanomas correlate with patient disease-specific survival using global gene expression profiling. Many of the identified potential markers of poor prognosis were associated with the epithelial-mesenchymal transition, extracellular matrix formation, and angiogenesis. We studied further the significance of one of the genes, prolyl 4-hydroxylase subunit alpha 1 (P4HA1), in melanoma progression. P4HA1 depletion in melanoma cells reduced cell adhesion, invasion, and viability in vitro. In melanoma xenograft assays, we found that P4HA1 knockdown reduced melanoma tumor invasion as well as the deposition of collagens, particularly type IV collagen, in the interstitial extracellular matrix and in the basement membranes of tumor blood vessels, leading to vessel wall rupture and hemorrhages. Further, P4HA1 knockdown reduced the secretion of collagen triple helix repeat containing 1 (CTHRC1), an important mediator of melanoma cell migration and invasion, in vitro and its deposition around tumor blood vessels in vivo. Taken together, P4HA1 is an interesting potential prognostic marker and therapeutic target in primary melanomas, influencing many aspects of melanoma tumor progression.

ARX788, a novel anti-HER2 antibody-drug conjugate, shows anti-tumor effects in preclinical models of trastuzumab emtansine-resistant HER2-positive breast cancer and gastric cancer.

The majority of HER2-positive breast or gastric cancers treated with T-DM1 eventually show resistance to this agent. We compared the effects of T-DM1 and ARX788, a novel anti-HER2 antibody-drug conjugate, on cell growth and apoptosis in HER2-positive breast cancer and gastric cancer cell lines sensitive to T-DM1, gastric cancer cell lines resistant to T-DM1, HER2-negative breast cancer cell lines, and T-DM1-resistant xenograft models. ARX788 was effective in T-DM1-resistant in vitro and in vivo models of HER2-positive breast cancer and gastric cancer. ARX788 showed a pronounced growth inhibitory effect on all five HER2-positive cell lines tested, of which two gastric cancer cell lines had acquired resistance to T-DM1. ARX788 evoked more apoptotic events compared to T-DM1. While JIMT-1 and RN-87 xenograft tumors progressed on T-DM1 treatment, all such tumors responded to ARX788, and four out of the six JIMT-1 tumors and nine out of the twelve RN-87 tumors disappeared during the ARX788 treatment. Mice treated with ARX788 survived longer than those treated with T-DM1. The data support evaluation of ARX788 in patients with HER2-positive breast cancer or gastric cancer including cancers that progress during T-DM1 therapy.

Monotherapy efficacy of blood-brain barrier permeable small molecule reactivators of protein phosphatase 2A in glioblastoma.

Glioblastoma is a fatal disease in which most targeted therapies have clinically failed. However, pharmacological reactivation of tumour suppressors has not been thoroughly studied as yet as a glioblastoma therapeutic strategy. Tumour suppressor protein phosphatase 2A is inhibited by non-genetic mechanisms in glioblastoma, and thus, it would be potentially amendable for therapeutic reactivation. Here, we demonstrate that small molecule activators of protein phosphatase 2A, NZ-8-061 and DBK-1154, effectively cross the in vitro model of blood-brain barrier, and in vivo partition to mouse brain tissue after oral dosing. In vitro, small molecule activators of protein phosphatase 2A exhibit robust cell-killing activity against five established glioblastoma cell lines, and nine patient-derived primary glioma cell lines. Collectively, these cell lines have heterogeneous genetic background, kinase inhibitor resistance profile and stemness properties; and they represent different clinical glioblastoma subtypes. Moreover, small molecule activators of protein phosphatase 2A were found to be superior to a range of kinase inhibitors in their capacity to kill patient-derived primary glioma cells. Oral dosing of either of the small molecule activators of protein phosphatase 2A significantly reduced growth of infiltrative intracranial glioblastoma tumours. DBK-1154, with both higher degree of brain/blood distribution, and more potent in vitro activity against all tested glioblastoma cell lines, also significantly increased survival of mice bearing orthotopic glioblastoma xenografts. In summary, this report presents a proof-of-principle data for blood-brain barrier-permeable tumour suppressor reactivation therapy for glioblastoma cells of heterogenous molecular background. These results also provide the first indications that protein phosphatase 2A reactivation might be able to challenge the current paradigm in glioblastoma therapies which has been strongly focused on targeting specific genetically altered cancer drivers with highly specific inhibitors. Based on demonstrated role for protein phosphatase 2A inhibition in glioblastoma cell drug resistance, small molecule activators of protein phosphatase 2A may prove to be beneficial in future glioblastoma combination therapies.

Predicting In Vivo Payloads Delivery using a Blood-brain Tumor-barrier in a Dish.

Highly selective by nature, the blood-brain barrier (BBB) is essential for the brain homeostasis in physiological conditions. However, in the context of brain tumors, the molecular selectivity of BBB also shields the neoplastic cells by blocking the delivery of peripherally administered chemotherapies. The development of novel drugs (including nanoparticles) targeting malignant brain tumors ideally requires the use of preclinical animal models to study the drug's transcytosis and antitumor efficacy. In order to comply with the 3R principle (refine, reduce, and replace) to reduce the number of laboratory animals in experimental setup and perform the high-throughput screening of a large library of antitumor agents, we developed a reproducible in vitro human and murine mimic of the blood-brain tumor-barrier (BBTB) using three-layered cultures of endothelial cells, astrocytes, and patient-derived glioblastoma spheres. For higher scalability and reproducibility, commercial cell lines or immortalized cells have been used in tailored conditions to allow the formation of a barrier resembling the actual BBB. Here we describe a protocol to obtain a BBTB mimic by culturing endothelial cells in contact with astrocytes at specific cell densities on inserts. This BBTB mimic can be used, for instance, for the quantification and confocal imaging of the nanoparticle passage through the endothelial and astrocytic barriers, in addition to the evaluation of the tumor cell targeting within the same assay. Moreover, we show that the obtained data can be used to predict the behavior of nanoparticles in preclinical animal models. In a broader perspective, this in vitro model could be adapted to other neurodegenerative diseases for the determination of the passage of new therapeutic molecules through the BBB and/or be supplemented with brain organoids to directly evaluate the efficacy of drugs.

A Novel Anti-HER2 Antibody-Drug Conjugate XMT-1522 for HER2-Positive Breast and Gastric Cancers Resistant to Trastuzumab Emtansine.

Most patients with HER2-positive breast or gastric cancer exhibit primary or acquired resistance to trastuzumab emtansine (T-DM1), and such patients may have limited therapeutic options. XMT-1522 is a novel anti-HER2 antibody-drug conjugate. We compared XMT-1522 to T-DM1 in preclinical models. The effects of XMT-1522 and T-DM1 on cell survival and apoptosis were compared in six HER2-positive breast cancer or gastric cancer cell lines, of which three lines were T-DM1-sensitive (N-87, OE-19, JIMT-1) and three T-DM1-resistant (RN-87, ROE-19, SNU-216). We compared these agents also in the HER2-negative breast cancer cell line MCF-7, and in mouse RN-87 and JIMT-1 xenograft models. Cell survival was assessed using the AlamarBlue method and apoptosis with the Caspase-Glo 3/7 method. XMT-1522 inhibited the growth of all six HER2-positive cell lines. The proportions of cells that survived XMT-1522 treatment were smaller as compared with T-DM1, particularly in the T-DM1-resistant cell lines. XMT-1522 induced more cell apoptosis compared with T-DM1. While RN-87 and JIMT-1 xenograft tumors progressed on T-DM1 treatment, all tumors responded to XMT-1522, and all but one tumor disappeared during the XMT-1522 treatment. XMT-1522 had a strong antitumor effect on RN-87 and JIMT-1 xenografts that progressed on T-DM1. We conclude that XMT-1522 was effective in HER2-positive breast cancer and gastric cancer cell lines resistant to T-DM1, and in xenograft models resistant to T-DM1. The results support the testing of XMT-1522 in clinical trials in patients with HER2-positive cancer.

Octreotide Conjugates for Tumor Targeting and Imaging.

Tumor targeting has emerged as an advantageous approach to improving the efficacy and safety of cytotoxic agents or radiolabeled ligands that do not preferentially accumulate in the tumor tissue. The somatostatin receptors (SSTRs) belong to the G-protein-coupled receptor superfamily and they are overexpressed in many neuroendocrine tumors (NETs). SSTRs can be efficiently targeted with octreotide, a cyclic octapeptide that is derived from native somatostatin. The conjugation of cargoes to octreotide represents an attractive approach for effective tumor targeting. In this study, we conjugated octreotide to cryptophycin, which is a highly cytotoxic depsipeptide, through the protease cleavable Val-Cit dipeptide linker using two different self-immolative moieties. The biological activity was investigated in vitro and the self-immolative part largely influenced the stability of the conjugates. Replacement of cryptophycin by the infrared cyanine dye Cy5.5 was exploited to elucidate the tumor targeting properties of the conjugates in vitro and in vivo. The compound efficiently and selectively internalized in cells overexpressing SSTR2 and accumulated in xenografts for a prolonged time. Our results on the in vivo properties indicate that octreotide may serve as an efficient delivery vehicle for tumor targeting.

Vulnerability of invasive glioblastoma cells to lysosomal membrane destabilization.

The current clinical care of glioblastomas leaves behind invasive, radio- and chemo-resistant cells. We recently identified mammary-derived growth inhibitor (MDGI/FABP3) as a biomarker for invasive gliomas. Here, we demonstrate a novel function for MDGI in the maintenance of lysosomal membrane integrity, thus rendering invasive glioma cells unexpectedly vulnerable to lysosomal membrane destabilization. MDGI silencing impaired trafficking of polyunsaturated fatty acids into cells resulting in significant alterations in the lipid composition of lysosomal membranes, and subsequent death of the patient-derived glioma cells via lysosomal membrane permeabilization (LMP). In a preclinical model, treatment of glioma-bearing mice with an antihistaminergic LMP-inducing drug efficiently eradicated invasive glioma cells and secondary tumours within the brain. This unexpected fragility of the aggressive infiltrating cells to LMP provides new opportunities for clinical interventions, such as re-positioning of an established antihistamine drug, to eradicate the inoperable, invasive, and chemo-resistant glioma cells from sustaining disease progression and recurrence.

Gene expression analyses of primary melanomas reveal CTHRC1 as an important player in melanoma progression.

Melanoma is notorious for its high tendency to metastasize and its refractoriness to conventional treatments after metastasis, and the responses to most targeted therapies are short-lived. A better understanding of the molecular mechanisms behind melanoma development and progression is needed to develop more effective therapies and to identify new markers to predict disease behavior. Here, we compared the gene expression profiles of benign nevi, and non-metastatic and metastatic primary melanomas to identify any common changes in disease progression. We identified several genes associated with inflammation, angiogenesis, and extracellular matrix modification to be upregulated in metastatic melanomas. We selected one of these genes, collagen triple helix repeat containing 1 (CTHRC1), for detailed analysis, and found that CTHRC1 was expressed in both melanoma cells and the associated fibroblasts, as well as in the endothelium of tumor blood vessels. Knockdown of CTHRC1 expression by shRNAs in melanoma cells inhibited their migration in Transwell assays and their invasion in three-dimensional collagen and Matrigel matrices. We also elucidated the possible down-stream effectors of CTHRC1 by gene expression profiling of the CTHRC1-knockdown cells. Our analyses showed that CTHRC1 is regulated coordinately with fibronectin and integrin ß3 by the pro-invasive and -angiogenic transcription factor NFATC2. We also found CTHRC1 to be a target of TFGß and BRAF. These data highlight the importance of tumor stroma in melanoma progression. Furthermore, CTHRC1 was recognized as an important mediator of melanoma cell migration and invasion, providing together with its regulators-NFATC2, TGFß, and BRAF-attractive therapeutic targets against metastatic melanomas.

Chemotactic G protein-coupled receptors control cell migration by repressing autophagosome biogenesis.

Chemotactic migration is a fundamental behavior of cells and its regulation is particularly relevant in physiological processes such as organogenesis and angiogenesis, as well as in pathological processes such as tumor metastasis. The majority of chemotactic stimuli activate cell surface receptors that belong to the G protein-coupled receptor (GPCR) superfamily. Although the autophagy machinery has been shown to play a role in cell migration, its mode of regulation by chemotactic GPCRs remains largely unexplored. We found that ligand-induced activation of 2 chemotactic GPCRs, the chemokine receptor CXCR4 and the urotensin 2 receptor UTS2R, triggers a marked reduction in the biogenesis of autophagosomes, in both HEK-293 and U87 glioblastoma cells. Chemotactic GPCRs exert their anti-autophagic effects through the activation of CAPNs, which prevent the formation of pre-autophagosomal vesicles from the plasma membrane. We further demonstrated that CXCR4- or UTS2R-induced inhibition of autophagy favors the formation of adhesion complexes to the extracellular matrix and is required for chemotactic migration. Altogether, our data reveal a new link between GPCR signaling and the autophagy machinery, and may help to envisage therapeutic strategies in pathological processes such as cancer cell invasion.

Selenoprotein T Deficiency Leads to Neurodevelopmental Abnormalities and Hyperactive Behavior in Mice.

Selenoprotein T (SelT) is a newly discovered thioredoxin-like protein, which is abundantly but transiently expressed in the neural lineage during brain ontogenesis. Because its physiological function in the brain remains unknown, we developed a conditional knockout mouse line (Nes-Cre/SelTfl/fl) in which SelT gene is specifically disrupted in nerve cells. At postnatal day 7 (P7), these mice exhibited reduced volume of different brain structures, including hippocampus, cerebellum, and cerebral cortex. This phenotype, which is observed early during the first postnatal week, culminated at P7 and was associated with increased loss of immature neurons but not glial cells, through apoptotic cell death. This phenomenon was accompanied by elevated levels of intracellular reactive oxygen species, which may explain the increased neuron demise and reduced brain structure volumes. At the second postnatal week, an increase in neurogenesis was observed in the cerebellum of Nes-Cre/SelTfl/fl mice, suggesting the occurrence of developmental compensatory mechanisms in the brain. In fact, the brain volume alterations observed at P7 were attenuated in adult mice. Nevertheless, SelT mutant mice exhibited a hyperactive behavior, suggesting that despite an apparent morphological compensation, SelT deficiency leads to cerebral malfunction in adulthood. Altogether, these results demonstrate that SelT exerts a neuroprotective role which is essential during brain development, and that its loss impairs mice behavior.