Lung cancer is the leading cause of cancer-related death worldwide. Here we demonstrate that elevated expression of dopamine and cyclic adenosine monophosphate-regulated phosphoprotein, Mr 32000 (DARPP-32), and its truncated splice variant t-DARPP promote lung tumor growth, while abrogation of DARPP-32 expression in human non-small-cell lung cancer (NSCLC) cells reduces tumor growth in orthotopic mouse models. We observe a physical interaction between DARPP-32 and inhibitory kappa B kinase-α (IKKα) that promotes NSCLC cell migration through non-canonical nuclear factor kappa-light-chain-enhancer of activated B cells 2 (NF-κB2) signaling. Bioinformatics analysis of 513 lung adenocarcinoma patients reveals that elevated t-DARPP isoform expression is associated with poor overall survival. Histopathological investigation of 62 human lung adenocarcinoma tissues also shows that t-DARPP expression is elevated with increasing tumor (T) stage. Our data suggest that DARPP-32 isoforms serve as a negative prognostic marker associated with increasing stages of NSCLC and may represent a novel therapeutic target.

In the last years, we have seen the emergence of different tools that have changed the face of biology from a simple modeling level to a more systematic science. The transparent zebrafish embryo is one of the living models in which, after germline transformation with reporter protein-coding genes, specific fluorescent cell populations can be followed at single-cell resolution. The genetically modified embryos, larvae and adults, resulting from the transformation, are individuals in which time lapse analysis, digital imaging quantification, FACS sorting and next-generation sequencing can be performed in specific times and tissues. These multifaceted genetic and cellular approaches have permitted to dissect molecular interactions at the subcellular, intercellular, tissue and whole-animal level, thus allowing integration of cellular and developmental genetics with molecular imaging in the resulting frame of modern biology. In this review, we describe a new step in the zebrafish road to system biology, based on the use of transgenic biosensor animals expressing fluorescent proteins under the control of signaling pathway-responsive cis-elements. In particular, we provide here the rationale and details of this powerful tool, trying to focus on its huge potentialities in basic and applied research, while also discussing limits and potential technological evolutions of this approach.

Zebrafish have proven to be a valuable model to study human cancer biology with the ultimate aim of developing new therapies. Danio rerio are amenable to in vivo imaging, high throughput drug screening, mutagenesis, and transgenesis, and they share histological and genetic similarities with Homo sapiens. The significance of zebrafish in the field of precision oncology is rapidly emerging. Indeed, modeling cancer in zebrafish has already been used to identify tumor biomarkers, define therapeutic targets and provide an in vivo platform for drug discovery. New zebrafish studies are starting to pave the way to direct individualized clinical applications. Patient-derived cancer cell xenograft models have demonstrated the feasibility of using zebrafish as a real time avatar of prognosis and drug response to identify the most ideal therapy for an individual patient. Genetic cancer modeling in zebrafish, now facilitated by rapidly evolving genome editing techniques, represents another innovative approach to recapitulate human oncogenesis and develop individualized treatments. Utilizing zebrafish to design customizable precision therapies will improve the clinical outcome of patients afflicted with cancer.

As effectors of the Hippo signaling cascade, YAP1 and TAZ are transcriptional regulators playing important roles in development, tissue homeostasis and cancer. A number of different cues, including mechanotransduction of extracellular stimuli, adhesion molecules, oncogenic signaling and metabolism modulate YAP1/TAZ nucleo-cytoplasmic shuttling. In the nucleus, YAP1/TAZ tether with the DNA binding proteins TEADs, to activate the expression of target genes that regulate proliferation, migration, cell plasticity, and cell fate. Based on responsive elements present in the human and zebrafish promoters of the YAP1/TAZ target gene CTGF, we established zebrafish fluorescent transgenic reporter lines of Yap1/Taz activity. These reporter lines provide an in vivo view of Yap1/Taz activity during development and adulthood at the whole organism level. Transgene expression was detected in many larval tissues including the otic vesicles, heart, pharyngeal arches, muscles and brain and is prominent in endothelial cells. Analysis of vascular development in yap1/taz zebrafish mutants revealed specific defects in posterior cardinal vein (PCV) formation, with altered expression of arterial/venous markers. The overactivation of Yap1/Taz in endothelial cells was sufficient to promote an aberrant vessel sprouting phenotype. Our findings confirm and extend the emerging role of Yap1/Taz in vascular development including angiogenesis.

Atherosclerosis, the major cause of myocardial infarction and stroke, results from converging inflammatory, metabolic, and biomechanical factors. Arterial lesions form at sites of low and disturbed blood flow but are suppressed by high laminar shear stress (LSS) mainly via transcriptional induction of the anti-inflammatory transcription factor, Kruppel-like factor 2 (Klf2). We therefore performed a whole genome CRISPR-Cas9 screen to identify genes required for LSS induction of Klf2. Subsequent mechanistic investigation revealed that LSS induces Klf2 via activation of both a MEKK2/3–MEK5–ERK5 kinase module and mitochondrial metabolism. Mitochondrial calcium and ROS signaling regulate assembly of a mitophagy- and p62-dependent scaffolding complex that amplifies MEKK–MEK5–ERK5 signaling. Blocking the mitochondrial pathway in vivo reduces expression of KLF2-dependent genes such as eNOS and inhibits vascular remodeling. Failure to activate the mitochondrial pathway limits Klf2 expression in regions of disturbed flow. This work thus defines a connection between metabolism and vascular inflammation that provides a new framework for understanding and developing treatments for vascular disease.

Metastasis is the major cause of mortality in prostate cancer patients. Factors such as genetic makeup and race play critical role in the outcome of therapies. This study was conducted to investigate the relevance of in metastatic prostate cancer disease in Caucasian and African-Americans. We employed race-specific prostate cancer models, clinical specimens, clinical data mining, gene-microarray, transcription-reporter assay, chromatin-immunoprecipitation (ChIP), IHC, transgenic-(tgfl/fl) zebrafish, and mouse metastasis models. BMI1 expression was observed to be elevated in metastatic tumors (lymph nodes, lungs, bones, liver) of Caucasian and African-American prostate cancer patients. The comparative analysis of stage III/IV tumors showed an increased BMI1 expression in African-Americans than Caucasians. TCGA and NIH/GEO clinical data corroborated to our findings. We show that expression (i) positively correlates to metastatic () and (ii) negative correlates to tumor suppressor () levels in tumors. The correlation was prominent in African-American tumors. We show that BMI1 regulates the transcriptional activation of , and We show the effect of pharmacological inhibition of BMI1 on the metastatic genome and invasiveness of tumor cells. Next, we show the anti-metastatic efficacy of BMI1-inhibitor in transgenic zebrafish and mouse metastasis models. Docetaxel as monotherapy has poor outcome on the growth of metastatic tumors. BMI1 inhibitor as an adjuvant improved the taxane therapy in race-based and models. BMI1, a major driver of metastasis, represents a promising therapeutic target for treating advanced prostate cancer in patients (including those belonging to high-risk group).

Dynamic modulation of endothelial cell-to-cell and cell–to–extracellular matrix (ECM) adhesion is essential for blood vessel patterning and functioning. Yet the molecular mechanisms involved in this process have not been completely deciphered. We identify the adhesion G protein–coupled receptor (ADGR) Latrophilin 2 (LPHN2) as a novel determinant of endothelial cell (EC) adhesion and barrier function. In cultured ECs, endogenous LPHN2 localizes at ECM contacts, signals through cAMP/Rap1, and inhibits focal adhesion (FA) formation and nuclear localization of YAP/TAZ transcriptional regulators, while promoting tight junction (TJ) assembly. ECs also express an endogenous LPHN2 ligand, fibronectin leucine-rich transmembrane 2 (FLRT2), that prevents ECM-elicited EC behaviors in an LPHN2-dependent manner. Vascular ECs of lphn2a knock-out zebrafish embryos become abnormally stretched, display a hyperactive YAP/TAZ pathway, and lack proper intercellular TJs. Consistently, blood vessels are hyperpermeable, and intravascularly injected cancer cells extravasate more easily in lphn2a null animals. Thus, LPHN2 ligands, such as FLRT2, may be therapeutically exploited to interfere with cancer metastatic dissemination.

Vascular permeability triggered by inflammation or ischemia promotes edema, exacerbates disease progression, and impairs tissue recovery. Vascular endothelial growth factor (VEGF) is a potent inducer of vascular permeability. VEGF plays an integral role in regulating vascular barrier function physiologically and in pathologies, including cancer, stroke, cardiovascular disease, retinal conditions, and COVID-19-associated pulmonary edema, sepsis, and acute lung injury. Understanding temporal molecular regulation of VEGF-induced vascular permeability will facilitate developing therapeutics to inhibit vascular permeability, while preserving tissue-restorative angiogenesis. Here, we demonstrate that VEGF signals through signal transducer and activator of transcription 3 (STAT3) to promote vascular permeability. We show that genetic STAT3 ablation reduces vascular permeability in STAT3-deficient endothelium of mice and VEGF-inducible zebrafish crossed with CRISPR/Cas9 generated Stat3 knockout zebrafish. Intercellular adhesion molecule 1 (ICAM-1) expression is transcriptionally regulated by STAT3 and VEGF-dependent STAT3 activation is regulated by JAK2. Pyrimethamine, an FDA-approved anti-microbial agent that inhibits STAT3-dependent transcription, substantially reduces VEGF-induced vascular permeability in zebrafish, mouse, and human endothelium. Collectively, our findings suggest that VEGF/VEGFR-2/JAK2/STAT3 signaling regulates vascular barrier integrity, and inhibition of STAT3-dependent activity reduces VEGF-induced vascular permeability.