Zebrafish polg2 knock-out recapitulates human POLG-disorders; implications for drug treatment.

The human mitochondrial DNA polymerase gamma is a holoenzyme, involved in mitochondrial DNA (mtDNA) replication and maintenance, composed of a catalytic subunit (POLG) and a dimeric accessory subunit (POLG2) conferring processivity. Mutations in POLG or POLG2 cause POLG-related diseases in humans, leading to a subset of Mendelian-inherited mitochondrial disorders characterized by mtDNA depletion (MDD) or accumulation of multiple deletions, presenting multi-organ defects and often leading to premature death at a young age. Considering the paucity of POLG2 models, we have generated a stable zebrafish polg2 mutant line (polg2ia304) by CRISPR/Cas9 technology, carrying a 10-nucleotide deletion with frameshift mutation and premature stop codon. Zebrafish polg2 homozygous mutants present slower development and decreased viability compared to wild type siblings, dying before the juvenile stage. Mutants display a set of POLG-related phenotypes comparable to the symptoms of human patients affected by POLG-related diseases, including remarkable MDD, altered mitochondrial network and dynamics, and reduced mitochondrial respiration. Histological analyses detected morphological alterations in high-energy demanding tissues, along with a significant disorganization of skeletal muscle fibres. Consistent with the last finding, locomotor assays highlighted a decreased larval motility. Of note, treatment with the Clofilium tosylate drug, previously shown to be effective in POLG models, could partially rescue MDD in Polg2 mutant animals. Altogether, our results point at zebrafish as an effective model to study the etiopathology of human POLG-related disorders linked to POLG2, and a suitable platform to screen the efficacy of POLG-directed drugs in POLG2-associated forms.

A novel DSP zebrafish model reveals training- and drug-induced modulation of arrhythmogenic cardiomyopathy phenotypes.

Arrhythmogenic cardiomyopathy (AC) is an inherited disorder characterized by progressive loss of the ventricular myocardium causing life-threatening ventricular arrhythmias, syncope and sudden cardiac death in young and athletes. About 40% of AC cases carry one or more mutations in genes encoding for desmosomal proteins, including Desmoplakin (Dsp). We present here the first stable Dsp knock-out (KO) zebrafish line able to model cardiac alterations and cell signalling dysregulation, characteristic of the AC disease, on which environmental factors and candidate drugs can be tested. Our stable Dsp knock-out (KO) zebrafish line was characterized by cardiac alterations, oedema and bradycardia at larval stages. Histological analysis of mutated adult hearts showed reduced contractile structures and abnormal shape of the ventricle, with thinning of the myocardial layer, vessels dilation and presence of adipocytes within the myocardium. Moreover, TEM analysis revealed "pale", disorganized and delocalized desmosomes. Intensive physical training protocol caused a global worsening of the cardiac phenotype, accelerating the progression of the disease. Of note, we detected a decrease of Wnt/ß-catenin signalling, recently associated with AC pathogenesis, as well as Hippo/YAP-TAZ and TGF-ß pathway dysregulation. Pharmacological treatment of mutated larvae with SB216763, a Wnt/ß-catenin agonist, rescued pathway expression and cardiac abnormalities, stabilizing the heart rhythm. Overall, our Dsp KO zebrafish line recapitulates many AC features observed in human patients, pointing at zebrafish as a suitable system for in vivo analysis of environmental modulators, such as the physical exercise, and the screening of pathway-targeted drugs, especially related to the Wnt/ß-catenin signalling cascade.

Anti-HER2 Super Stealth Immunoliposomes for Targeted-Chemotherapy.

Liposomes play an important role in the field of drug delivery by virtue of their biocompatibility and versatility as carriers. Stealth liposomes, obtained by surface decoration with hydrophilic polyethylene glycol (PEG) molecules, represent an important turning point in liposome technology, leading to significant improvements in the pharmacokinetic profile compared to naked liposomes. Nevertheless, the generation of effective targeted liposomes-a central issue for cancer therapy-has faced several difficulties and clinical phase failures. Active targeting remains a challenge for liposomes. In this direction, a new Super Stealth Immunoliposomes (SSIL2) composed of a PEG-bi-phospholipids derivative is designed that stabilizes the polymer shielding over the liposomes. Furthermore, its counterpart, conjugated to the fragment antigen-binding of trastuzumab (Fab'TRZ -PEG-bi-phospholipids), is firmly anchored on the liposomes surface and correctly orients outward the targeting moiety. Throughout this study, the performances of SSIL2 are evaluated and compared to classic stealth liposomes and stealth immunoliposomes in vitro in a panel of cell lines and in vivo studies in zebrafish larvae and rodent models. Overall, SSIL2 shows superior in vitro and in vivo outcomes, both in terms of safety and anticancer efficacy, thus representing a step forward in targeted cancer therapy, and valuable for future development.

STAT3 and HIF1α cooperatively mediate the transcriptional and physiological responses to hypoxia.

STAT3 and HIF1α are two fundamental transcription factors involved in many merging processes, like angiogenesis, metabolism, and cell differentiation. Notably, under pathological conditions, the two factors have been shown to interact genetically, but both the molecular mechanisms underlying such interactions and their relevance under physiological conditions remain unclear. In mouse embryonic stem cells (ESCs) we manage to determine the specific subset of hypoxia-induced genes that need STAT3 to be properly transcribed and, among them, fundamental genes like Vegfa, Hk1, Hk2, Pfkp and Hilpda are worth mentioning. Unexpectedly, we also demonstrated that the absence of STAT3 does not affect the expression of Hif1α mRNA nor the stabilization of HIF1α protein, but the STAT3-driven regulation of the hypoxia-dependent subset of gene could rely on the physical interaction between STAT3 and HIF1α. To further elucidate the physiological roles of this STAT3 non-canonical nuclear activity, we used a CRISPR/Cas9 zebrafish stat3 knock-out line. Notably, hypoxia-related fluorescence of the hypoxia zebrafish reporter line (HRE:mCherry) cannot be induced when Stat3 is not active and, while Stat3 Y705 phosphorylation seems to have a pivotal role in this process, S727 does not affect the Stat3-dependent hypoxia response. Hypoxia is fundamental for vascularization, angiogenesis and immune cells mobilization; all processes that, surprisingly, cannot be induced by low oxygen levels when Stat3 is genetically ablated. All in all, here we report the specific STAT3/HIF1α-dependent subset of genes in vitro and, for the first time with an in vivo model, we determined some of the physiological roles of STAT3-hypoxia crosstalk.

The Saccharomyces cerevisiae mitochondrial DNA polymerase and its contribution to the knowledge about human POLG-related disorders.

Most eukaryotes possess a mitochondrial genome, called mtDNA. In animals and fungi, the replication of mtDNA is entrusted by the DNA polymerase γ, or Pol γ. The yeast Pol γ is composed only of a catalytic subunit encoded by MIP1. In humans, Pol γ is a heterotrimer composed of a catalytic subunit homolog to Mip1, encoded by POLG, and two accessory subunits. In the last 25 years, more than 300 pathological mutations in POLG have been identified as the cause of several mitochondrial diseases, called POLG-related disorders, which are characterized by multiple mtDNA deletions and/or depletion in affected tissues. In this review, at first, we summarize the biochemical properties of yeast Mip1, and how mutations, especially those introduced recently in the N-terminal and C-terminal regions of the enzyme, affect the in vitro activity of the enzyme and the in vivo phenotype connected to the mtDNA stability and to the mtDNA extended and point mutability. Then, we focus on the use of yeast harboring Mip1 mutations equivalent to the human ones to confirm their pathogenicity, identify the phenotypic defects caused by these mutations, and find both mechanisms and molecular compounds able to rescue the detrimental phenotype. A closing chapter will be dedicated to other polymerases found in yeast mitochondria, namely Pol ζ, Rev1 and Pol η, and to their genetic interactions with Mip1 necessary to maintain mtDNA stability and to avoid the accumulation of spontaneous or induced point mutations.

CD271 activation prevents low to high-risk progression of cutaneous squamous cell carcinoma and improves therapy outcomes.

BACKGROUND: Cutaneous squamous cell carcinoma (cSCC) is the second most prevalent form of skin cancer, showing a rapid increasing incidence worldwide. Although most cSCC can be cured by surgery, a sizeable number of cases are diagnosed at advanced stages, with local invasion and distant metastatic lesions. In the skin, neurotrophins (NTs) and their receptors (CD271 and Trk) form a complex network regulating epidermal homeostasis. Recently, several works suggested a significant implication of NT receptors in cancer. However, CD271 functions in epithelial tumors are controversial and its precise role in cSCC is still to be defined. METHODS: Spheroids from cSCC patients with low-risk (In situ or Well-Differentiated cSCC) or high-risk tumors (Moderately/Poorly Differentiated cSCC), were established to explore histological features, proliferation, invasion abilities, and molecular pathways modulated in response to CD271 overexpression or activation in vitro. The effect of CD271 activities on the response to therapeutics was also investigated. The impact on the metastatic process and inflammation was explored in vivo and in vitro, by using zebrafish xenograft and 2D/3D models. RESULTS: Our data proved that CD271 is upregulated in Well-Differentiated tumors as compared to the more aggressive Moderately/Poorly Differentiated cSCC, both in vivo and in vitro. We demonstrated that CD271 activities reduce proliferation and malignancy marker expression in patient-derived cSCC spheroids at each tumor grade, by increasing neoplastic cell differentiation. CD271 overexpression significantly increases cSCC spheroid mass density, while it reduces their weight and diameter, and promotes a major fold-enrichment in differentiation and keratinization genes. Moreover, both CD271 overexpression and activation decrease cSCC cell invasiveness in vitro. A significant inhibition of the metastatic process by CD271 was observed in a newly established zebrafish cSCC model. We found that the recruitment of leucocytes by CD271-overexpressing cells directly correlates with tumor killing and this finding was further highlighted by monocyte infiltration in a THP-1-SCC13 3D model. Finally, CD271 activity synergizes with Trk receptor inhibition, by reducing spheroid viability, and significantly improves the outcome of photodynamic therapy (PTD) or chemotherapy in spheroids and zebrafish. CONCLUSION: Our study provides evidence that CD271 could prevent the switch between low to high-risk cSCC tumors. Because CD271 contributes to maintaining active differentiative paths and favors the response to therapies, it might be a promising target for future pharmaceutical development.

Deficiency of AP1 Complex Ap1g1 in Zebrafish Model Led to Perturbation of Neurodevelopment, Female and Male Fertility; New Insight to Understand Adaptinopathies.

In vertebrates, two homologous heterotetrameric AP1 complexes regulate the intracellular protein sorting via vesicles. AP-1 complexes are ubiquitously expressed and are composed of four different subunits: γ, ß1, µ1 and σ1. Two different complexes are present in eukaryotic cells, AP1G1 (contains γ1 subunit) and AP1G2 (contains γ2 subunit); both are indispensable for development. One additional tissue-specific isoform exists for µ1A, the polarized epithelial cells specific to µ1B; two additional tissue-specific isoforms exist for σ1A: σ1B and σ1C. Both AP1 complexes fulfil specific functions at the trans-Golgi network and endosomes. The use of different animal models demonstrated their crucial role in the development of multicellular organisms and the specification of neuronal and epithelial cells. Ap1g1 (γ1) knockout mice cease development at the blastocyst stage, while Ap1m1 (µ1A) knockouts cease during mid-organogenesis. A growing number of human diseases have been associated with mutations in genes encoding for the subunits of adaptor protein complexes. Recently, a new class of neurocutaneous and neurometabolic disorders affecting intracellular vesicular traffic have been referred to as adaptinopathies. To better understand the functional role of AP1G1 in adaptinopathies, we generated a zebrafish ap1g1 knockout using CRISPR/Cas9 genome editing. Zebrafish ap1g1 knockout embryos cease their development at the blastula stage. Interestingly, heterozygous females and males have reduced fertility and showed morphological alterations in the brain, gonads and intestinal epithelium. An analysis of mRNA profiles of different marker proteins and altered tissue morphologies revealed dysregulated cadherin-mediated cell adhesion. These data demonstrate that the zebrafish model organism enables us to study the molecular details of adaptinopathies and thus also develop treatment strategies.

The mitochondrial inhibitor IF1 binds to the ATP synthase OSCP subunit and protects cancer cells from apoptosis.

The mitochondrial protein IF1 binds to the catalytic domain of the ATP synthase and inhibits ATP hydrolysis in ischemic tissues. Moreover, IF1 is overexpressed in many tumors and has been shown to act as a pro-oncogenic protein, although its mechanism of action is still debated. Here, we show that ATP5IF1 gene disruption in HeLa cells decreases colony formation in soft agar and tumor mass development in xenografts, underlining the role of IF1 in cancer. Notably, the lack of IF1 does not affect proliferation or oligomycin-sensitive mitochondrial respiration, but it sensitizes the cells to the opening of the permeability transition pore (PTP). Immunoprecipitation and proximity ligation analysis show that IF1 binds to the ATP synthase OSCP subunit in HeLa cells under oxidative phosphorylation conditions. The IF1-OSCP interaction is confirmed by NMR spectroscopy analysis of the recombinant soluble proteins. Overall, our results suggest that the IF1-OSCP interaction protects cancer cells from PTP-dependent apoptosis under normoxic conditions.

Hyperactivation of Wnt/ß-catenin and Jak/Stat3 pathways in human and zebrafish foetal growth restriction models: Implications for pharmacological rescue.

Foetal Growth Restriction (FGR), previously known as Intrauterine Growth Restriction (IUGR), is an obstetrical condition due to placental insufficiency, affecting yearly about 30 million newborns worldwide. In this work, we aimed to identify and pharmacologically target signalling pathways specifically involved in the FGR condition, focusing on FGR-related cardiovascular phenotypes. The transcriptional profile of human umbilical cords from FGR and control cases was compared with the response to hypoxia of zebrafish (Danio rerio) transgenic lines reporting in vivo the activity of twelve signalling pathways involved in embryonic development. Wnt/ß-catenin and Jak/Stat3 were found as key pathways significantly dysregulated in both human and zebrafish samples. This information was used in a chemical-genetic analysis to test drugs targeting Wnt/ß-catenin and Jak/Stat3 pathways to rescue a set of FGR phenotypes, including growth restriction and cardiovascular modifications. Treatments with the Wnt/ß-catenin agonist SB216763 successfully rescued body dimensions, cardiac shape, and vessel organization in zebrafish FGR models. Our data support the Wnt/ß-catenin pathway as a key FGR marker and a promising target for pharmacological intervention in the FGR condition.

The Zebrafish model in dermatology: an update for clinicians.

Recently, the zebrafish has been established as one of the most important model organisms for medical research. Several studies have proved that there is a high level of similarity between human and zebrafish genomes, which encourages the use of zebrafish as a model for understanding human genetic disorders, including cancer. Interestingly, zebrafish skin shows several similarities to human skin, suggesting that this model organism is particularly suitable for the study of neoplastic and inflammatory skin disorders. This paper appraises the specific characteristics of zebrafish skin and describes the major applications of the zebrafish model in dermatological research.

Multimodal Characterization of Seizures in Zebrafish Larvae.

Epilepsy accounts for a significant proportion of the world's disease burden. Indeed, many research efforts are produced both to investigate the basic mechanism ruling its genesis and to find more effective therapies. In this framework, the use of zebrafish larvae, owing to their peculiar features, offers a great opportunity. Here, we employ transgenic zebrafish larvae expressing GCaMP6s in all neurons to characterize functional alterations occurring during seizures induced by pentylenetetrazole. Using a custom two-photon light-sheet microscope, we perform fast volumetric functional imaging of the entire larval brain, investigating how different brain regions contribute to seizure onset and propagation. Moreover, employing a custom behavioral tracking system, we outline the progressive alteration of larval swim kinematics, resulting from different grades of seizures. Collectively, our results show that the epileptic larval brain undergoes transitions between diverse neuronal activity regimes. Moreover, we observe that different brain regions are progressively recruited into the generation of seizures of diverse severity. We demonstrate that midbrain regions exhibit highest susceptibility to the convulsant effects and that, during periods preceding abrupt hypersynchronous paroxysmal activity, they show a consistent increase in functional connectivity. These aspects, coupled with the hub-like role that these regions exert, represent important cues in their identification as epileptogenic hubs.

Notch controls the cell cycle to define leader versus follower identities during collective cell migration.

Coordination of cell proliferation and migration is fundamental for life, and its dysregulation has catastrophic consequences, such as cancer. How cell cycle progression affects migration, and vice versa, remains largely unknown. We address these questions by combining in silico modelling and in vivo experimentation in the zebrafish trunk neural crest (TNC). TNC migrate collectively, forming chains with a leader cell directing the movement of trailing followers. We show that the acquisition of migratory identity is autonomously controlled by Notch signalling in TNC. High Notch activity defines leaders, while low Notch determines followers. Moreover, cell cycle progression is required for TNC migration and is regulated by Notch. Cells with low Notch activity stay longer in G1 and become followers, while leaders with high Notch activity quickly undergo G1/S transition and remain in S-phase longer. In conclusion, TNC migratory identities are defined through the interaction of Notch signalling and cell cycle progression.

Fast whole-brain imaging of seizures in zebrafish larvae by two-photon light-sheet microscopy.

Light-sheet fluorescence microscopy (LSFM) enables real-time whole-brain functional imaging in zebrafish larvae. Conventional one-photon LSFM can however induce undesirable visual stimulation due to the use of visible excitation light. The use of two-photon (2P) excitation, employing near-infrared invisible light, provides unbiased investigation of neuronal circuit dynamics. However, due to the low efficiency of the 2P absorption process, the imaging speed of this technique is typically limited by the signal-to-noise-ratio. Here, we describe a 2P LSFM setup designed for non-invasive imaging that enables quintuplicating state-of-the-art volumetric acquisition rate of the larval zebrafish brain (5 Hz) while keeping low the laser intensity on the specimen. We applied our system to the study of pharmacologically-induced acute seizures, characterizing the spatial-temporal dynamics of pathological activity and describing for the first time the appearance of caudo-rostral ictal waves (CRIWs).

An adaptive registration algorithm for zebrafish larval brain images.

BACKGROUND AND OBJECTIVE: Zebrafish (Danio rerio) in their larval stages have grown increasingly popular as excellent vertebrate models for neurobiological research. Researchers can apply various tools in order to decode the neural structure patterns which can aid the understanding of vertebrate brain development. In order to do so, it is essential to map the gene expression patterns to an anatomical reference precisely. However, high accuracy in sample registration is sometimes difficult to achieve due to laboratory- or protocol-dependent variabilities. METHODS: In this paper, we propose an accurate adaptive registration algorithm for volumetric zebrafish larval image datasets using a synergistic combination of attractive Free-Form-Deformation (FFD) and diffusive Demons algorithms. A coarse registration is achieved first for 3D volumetric data using a 3D affine transformation. A localized registration algorithm in form of a B-splines based FFD is applied next on the coarsely registered volume. Finally, the Demons algorithm is applied on this FFD registered volume for achieving fine registration by making the solution noise resilient. RESULTS: Results Experimental procedures are carried out on a number of 72 hpf (hours post fertilization) 3D confocal zebrafish larval datasets. Comparisons with state-of-the-art methods including some ablation studies clearly demonstrate the effectiveness of the proposed method. CONCLUSIONS: Our adaptive registration algorithm significantly aids Zebrafish imaging analysis over current methods for gene expression anatomical mapping, such as Vibe-Z. We believe the proposed solution would be able to overcome the requirement of high quality images which currently limits the applicability of Zebrafish in neuroimaging research.

Macrophage-Mediated Melanoma Reduction after HP-NAP Treatment in a Zebrafish Xenograft Model.

The Helicobacter pylori Neutrophil Activating Protein (HP-NAP) is endowed with immunomodulatory properties that make it a potential candidate for anticancer therapeutic applications. By activating cytotoxic Th1 responses, HP-NAP inhibits the growth of bladder cancer and enhances the anti-tumor activity of oncolytic viruses in the treatment of metastatic breast cancer and neuroendocrine tumors. The possibility that HP-NAP exerts its anti-tumor effect also by modulating the activity of innate immune cells has not yet been explored. Taking advantage of the zebrafish model, we examined the therapeutic efficacy of HP-NAP against metastatic human melanoma, limiting the observational window to 9 days post-fertilization, well before the maturation of the adaptive immunity. Human melanoma cells were xenotransplanted into zebrafish embryos and tracked in the presence or absence of HP-NAP. The behavior and phenotype of macrophages and the impact of their drug-induced depletion were analyzed exploiting macrophage-expressed transgenes. HP-NAP administration efficiently inhibited tumor growth and metastasis and this was accompanied by strong recruitment of macrophages with a pro-inflammatory profile at the tumor site. The depletion of macrophages almost completely abrogated the ability of HP-NAP to counteract tumor growth. Our findings highlight the pivotal role of activated macrophages in counteracting melanoma growth and support the notion that HP-NAP might become a new biological therapeutic agent for the treatment of metastatic melanomas.

Biodegradable nanoparticles combining cancer cell targeting and anti-angiogenic activity for synergistic chemotherapy in epithelial cancer.

A biodegradable engineered nanoplatform combining anti-angiogenic activity and targeting of cancer cells to improve the anticancer activity of docetaxel (DTX) is here proposed. Indeed, we have developed biodegradable nanoparticles (NPs) of poly(ethylene glycol)-poly(ε-caprolactone), exposing on the surface both folate motifs (Fol) for recognition in cells overexpressing Folate receptor-α (FRα) and the anti-angiogenic hexapeptide aFLT1. NPs showed a size around 100 nm, the exposure of 60% of Fol moieties on the surface, and the ability to entrap DTX and sustain its release with time. NPs were stable in simulated biological fluids and slightly interacted with Fetal Bovine serum, especially in the formulation decorated with Fol and aFLT1. The presence of Fol on NPs did not impair the anti-angiogenic activity of aFLT1, as assessed by in vitro tube formation assay in HUVEC endothelial cells. In both 2D and 3D KB cell cultures in vitro, the cytotoxicity of DTX loaded in NPs was not significantly affected by Fol/aFLT1 double decoration compared to free DTX. Remarkably, NPs distributed differently in 3D multicellular spheroids of FRα-positive KB cancer cells depending on the type of ligand displayed on the surface. In particular, NPs unmodified on the surface were randomly distributed in the spheroid, whereas the presence of Fol promoted the accumulation in the outer rims of the spheroid. Finally, NPs with Fol and aFLT1 gave a uniform distribution throughout the spheroid structure. When tested in zebrafish embryos xenografted with KB cells, NPs displaying Fol/aFLT1 reduced DTX systemic toxicity and inhibited the growth of the tumor mass and associated vasculature synergistically. Overall, nanotechnology offers excellent ground for combining therapeutic concepts in cancer, paving the way to novel multifunctional nanopharmaceuticals decorated with bioactive elements that can significantly improve therapeutic outcomes.

Activation of cGMP-Dependent Protein Kinase Restricts Melanoma Growth and Invasion by Interfering with the EGF/EGFR Pathway.

Drug resistance mechanisms still characterize metastatic melanoma, despite the new treatments that have been recently developed. Targeting of the cGMP/protein kinase G pathway is emerging as a therapeutic approach in cancer research. In this study, we evaluated the anticancer effects of two polymeric-linked dimeric cGMP analogs able to bind and activate protein kinase G, called protein kinase G activators (PAs) 4 and 5. PA5 was identified as the most effective compound on melanoma cell lines as well as on patient-derived metastatic melanoma cells cultured as three-dimensional spheroids and in a zebrafish melanoma model. PA5 was able to significantly reduce cell viability, size, and invasion of melanoma spheroids. Importantly, PA5 showed a tumor-specific outcome because no toxic effect was observed in healthy melanocytes exposed to the cGMP analog. We defined that by triggering protein kinase G, PA5 interfered with the EGF pathway as shown by lower EGFR phosphorylation and reduction of activated, phosphorylated forms of protein kinase B and extracellular signal‒regulated kinase 1/2 in melanoma cells. Finally, PA5 significantly reduced the metastatic process in zebrafish. These studies open future perspectives for the cGMP analog PA5 as a potential therapeutic strategy for melanoma.

STK11 Prevents Invasion through Signal Transducer and Activator of Transcription 3/5 and FAK Repression in Cutaneous Melanoma.

The STK11/LKB1 is a tumor suppressor involved in metabolism and cell motility. In BRAFV600E melanoma, STK11 is inactivated by extracellular signal‒regulated kinase and RSK, preventing it from binding and activating adenosine monophosphate-activated protein kinase and promoting melanoma cell proliferation. Although STK11 mutations occur in 5‒10% of cutaneous melanoma, few functional studies have been performed. By knocking out STK11 with CRISPR/Cas9 in two human BRAF-mutant melanoma cell lines, we found that STK11 loss reduced the sensitivity to a BRAF inhibitor. More strikingly, STK11 loss led to an increased invasive phenotype in both three-dimensional spheroids and in vivo zebrafish xenograft models. STK11 overexpression consistently reversed the invasive phenotype. Interestingly, STK11 knockout increased invasion also in an NRAS-mutant melanoma cell line. Furthermore, although STK11 was expressed in primary human melanoma tumors, its expression significantly decreased in melanoma metastases, especially in brain metastases. In the STK11-knockout cells, we observed increased activating phosphorylation of signal transducer and activator of transcription 3/5 and FAK. Using inhibitors of signal transducer and activator of transcription 3/5 and FAK, we reversed the invasive phenotype in both BRAF- and NRAS-mutated cells. Our findings confirm an increased invasive phenotype on STK11 inactivation in BRAF- and NRAS-mutant cutaneous melanoma that can be targeted by signal transducer and activator of transcription 3/5 and FAK inhibition.

Specific Activation of the CD271 Intracellular Domain in Combination with Chemotherapy or Targeted Therapy Inhibits Melanoma Progression.

CD271 (NGFR) is a neurotrophin receptor that belongs to the tumor necrosis receptor (TNFR) family. Upon ligand binding, CD271 can mediate either survival or cell death. Although the role of CD271 as a marker of tumor-initiating cells is still a matter of debate, its role in melanoma progression has been well documented. Moreover, CD271 has been shown to be upregulated after exposure to both chemotherapy and targeted therapy. In this study, we demonstrate that activation of CD271 by a short ß-amyloid-derived peptide (Aß(25-35)) in combination with either chemotherapy or MAPK inhibitors induces apoptosis in 2D and 3D cultures of eight melanoma cell lines. This combinatorial treatment significantly reduced metastasis in a zebrafish xenograft model and led to significantly decreased tumor volume in mice. Administration of Aß(25-35) in ex vivo tumors from immunotherapy- and targeted therapy-resistant patients significantly reduced proliferation of melanoma cells, showing that activation of CD271 can overcome drug resistance. Aß(25-35) was specific to CD271-expressing cells and induced CD271 cleavage and phosphorylation of JNK (pJNK). The direct protein-protein interaction of pJNK with CD271 led to PARP1 cleavage, p53 and caspase activation, and pJNK-dependent cell death. Aß(25-35) also mediated mitochondrial reactive oxygen species (mROS) accumulation, which induced CD271 overexpression. Finally, CD271 upregulation inhibited mROS production, revealing the presence of a negative feedback loop in mROS regulation. These results indicate that targeting CD271 can activate cell death pathways to inhibit melanoma progression and potentially overcome resistance to targeted therapy. SIGNIFICANCE: The discovery of a means to specifically activate the CD271 death domain reveals unknown pathways mediated by the receptor and highlights new treatment possibilities for melanoma.

Y705 and S727 are required for the mitochondrial import and transcriptional activities of STAT3, and for regulation of stem cell proliferation.

The STAT3 transcription factor, acting both in the nucleus and mitochondria, maintains embryonic stem cell pluripotency and promotes their proliferation. In this work, using zebrafish, we determined in vivo that mitochondrial STAT3 regulates mtDNA transcription in embryonic and larval stem cell niches and that this activity affects their proliferation rates. As a result, we demonstrated that import of STAT3 inside mitochondria requires Y705 phosphorylation by Jak, whereas its mitochondrial transcriptional activity, as well as its effect on proliferation, depends on the MAPK target S727. These data were confirmed using mouse embryonic stem cells: although the Y705-mutated STAT3 cannot enter mitochondria, the S727 mutation does not affect import into the organelle and is responsible for STAT3-dependent mitochondrial transcription. Surprisingly, STAT3-dependent increase of mitochondrial transcription appears to be independent from STAT3 binding to STAT3-responsive elements. Finally, loss-of-function experiments, with chemical inhibition of the JAK/STAT3 pathway or genetic ablation of stat3 gene, demonstrated that STAT3 is also required for cell proliferation in the intestine of zebrafish.