Focal adhesions (FAs) are critical multi-protein complexes regulating cell adhesion, migration, and survival, and their dysregulation contributes to cancer metastasis and vascular diseases. Despite extensive research on FA formation, little is known about FA turnover, particularly its regulation by autophagy. This study introduces a novel tandem fluorescence reporter capable of tracking the entire FA-phagy flux, from autophagosome formation to lysosomal degradation. The reporter, based on a red-green fluorescence system with a lysosome-specific cleavage site, integrates seamlessly into endogenous focal adhesion complexes, demonstrating sensitivity and specificity to autophagy stimuli. Validated in multiple cell lines, the tool revealed dynamic FA-phagy responses to starvation-induced autophagy and the involvement of autophagy regulators such as mTOR and ATG genes. This versatile reporter provides a powerful tool for investigating FA-phagy mechanisms, with significant implications for cancer biology and vascular research.

Despite the clinical use of anti-vascular endothelial growth factor (VEGF) antibodies (AVAs) in cancer therapy, resistance frequently develops, leading to disease progression. To address this, we identify a previously unknown role for breast cancer type 1 susceptibility protein (BRCA1)-associated RING domain 1 (BARD1) in modulating AVA sensitivity. Epigenetic modulation-via global and targeted DNA methylation-reveals BARD1 as a key regulator of angiogenesis. Sequential treatment with azacytidine overcomes AVA resistance in vivo. To enable precise epigenetic reactivation, we develop a liposomal CRISPR-deactivated Cas9 (dCas9)-TET1 system guided by BARD1-targeting single-guide RNAs (sgRNAs). This platform achieves CpG-specific demethylation of the BARD1 promoter, restores expression, and enhances AVA response. Additionally, BARD1 restoration, through either dCas9-TET1 or small interfering RNA (siRNA), significantly reduces tumor growth in combination with AVA in ovarian cancer models. These findings uncover a previously unrecognized function of BARD1 in tumor angiogenesis and demonstrate the potential of gene-specific epigenetic targeting to overcome AVA resistance.

Mechanical force generated by blood flow stimulates emergence of the first hematopoietic stem cells (HSCs) that populate the blood system. Force drives the transition of HSC precursors from an endothelial to hematopoietic identity, yet the molecular regulation of this fate switch remains poorly understood. We report that shear stress triggers adaptation in mitochondrial composition, ultrastructure, and function, which are essential for hematopoietic fate and engraftment potential. Shear stress remodels mitochondria in hemogenic endothelium by promoting mitochondrial gene transcription and protein synthesis. Laminar flow selectively initiates translation of 5' terminal polypyrimidine (5'TOP) motif-containing transcripts, which commonly encode ribosome and translation machinery. Flow-responsive metabolic reprogramming depends upon mechanistic target of rapamycin (mTOR) activation and is stymied when ribosome activity or mTOR is blocked. Conversely, chemical induction of mTOR mimics the effects of force on mitochondria and blood reconstituting potential and also partially rescues hematopoiesis in heartbeat mutants in utero. These findings identify mechanometabolism as a determinant of hematopoietic fate that could inform engineering of HSCs for disease modeling and treatment.

Supplementary Legends for Figures 1-3 and Tables 1-5, Methods from The miR-17/92 Polycistron Is Up-regulated in Sonic Hedgehog–Driven Medulloblastomas and Induced by N-myc in Sonic Hedgehog–Treated Cerebellar Neural Precursors

MiRNAs play crucial roles in a broad spectrum of biological processes, both physiological and pathological. Different reports implicate miR-205 in the control of breast stem cell properties. Differential miR-205 expression has been observed in different stages of mammary gland development and maturation. However, a functional role in this process has not been clearly demonstrated. We generated an miR-205 knockout in the FVB/N mouse strain, which is viable and characterized by enhanced mammary gland development. Indeed, mammary glands of miR-205-/- female mice at different ages (1.5 and 5.5 months) show increased outgrowth and branching. This evidence is consistent with our previously reported data demonstrating the direct miR-205-mediated targeting of HER3, a master regulator of mammary gland development, and the oncosuppressive activity of this microRNA in different types of breast cancer.

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Supplementary Table 2 from The miR-17/92 Polycistron Is Up-regulated in Sonic Hedgehog–Driven Medulloblastomas and Induced by N-myc in Sonic Hedgehog–Treated Cerebellar Neural Precursors

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Lymphatic drainage generates force that induces prostate cancer cell motility via activation of Yes-associated protein (YAP), but whether this response to fluid force is conserved across cancer types is unclear. Here, we show that shear stress corresponding to fluid flow in the initial lymphatics modifies taxis in breast cancer, whereas some cell lines use rapid amoeboid migration behavior in response to fluid flow, a separate subset decrease movement. Positive responders displayed transcriptional profiles characteristic of an amoeboid cell state, which is typical of cells advancing at the edges of neoplastic tumors. Regulation of the HIPPO tumor suppressor pathway and YAP activity also differed between breast subsets and prostate cancer. Although subcellular localization of YAP to the nucleus positively correlated with overall velocity of locomotion, YAP gain- and loss-of-function demonstrates that YAP inhibits breast cancer motility but is outcompeted by other pro-taxis mediators in the context of flow. Specifically, we show that RhoA dictates response to flow. GTPase activity of RhoA, but not Rac1 or Cdc42 Rho family GTPases, is elevated in cells that positively respond to flow and is unchanged in cells that decelerate under flow. Disruption of RhoA or the RhoA effector, Rho-associated kinase (ROCK), blocked shear stress-induced motility. Collectively, these findings identify biomechanical force as a regulator amoeboid cell migration and demonstrate stratification of breast cancer subsets by flow-sensing mechanotransduction pathways.

Podosomes and tight junctions (TJs) are subcellular compartments that both exist in endothelial cells and localize at cell surfaces. In contrast to the well-characterized role of TJs in maintaining cerebrovascular integrity, the specific function of endothelial podosomes remains unknown. Intriguingly, we discovered cross-talk between podosomes and TJs in human brain endothelial cells. Tight junction scaffold proteins ZO-1 and ZO-2 localize at podosomes in response to phorbol-12-myristate-13-acetate treatment. We found that both ZO proteins are essential for podosome formation and function. Rather than being derived from new protein synthesis, podosomal ZO-1 and ZO-2 are relocated from a pre-existing pool found at the peripheral plasma membrane with enhanced physical interaction with cortactin, a known protein marker for podosomes. Sequestration of ZO proteins in podosomes weakens tight junction complex formation, leading to increased endothelial cell permeability. This effect can be further attenuated by podosome inhibitor PP2. Altogether, our data revealed a novel cellular function of podosomes, specifically, their ability to negatively regulate tight junction and endothelial barrier integrity, which have been linked to a variety of cerebrovascular diseases.

BackgroundDNA methylation inhibitors are logical therapeutic candidates for ependymomas originating in the posterior fossa of the brain. Our objective was to test the safety of infusing 5-Azacytidine (5-AZA), a DNA methylation inhibitor, directly into cerebrospinal fluid (CSF) spaces of the fourth ventricle or tumor resection cavity in children with recurrent ependymoma originating in the posterior fossa.Materials and methodsIn patients with recurrent ependymoma whose disease originated in the posterior fossa, a maximal safe subtotal tumor resection was performed. At the conclusion of the tumor resection, a catheter was surgically placed into the fourth ventricle or tumor resection cavity and attached to a ventricular access device. CSF flow from the posterior fossa to the sacrum was confirmed by CINE phase contrast magnetic resonance imaging (MRI) postoperatively. 12 consecutive weekly 10milligram (mg) infusions of 5-Azacytidine (AZA) were planned. Disease response was monitored with MRI scans and CSF cytology.ResultsSix patients were enrolled. One patient was withdrawn prior to planned 5-AZA infusions due to surgical complications after tumor resection. The remaining five patients received 8, 12, 12, 12, and 12 infusions, respectively. There were no serious adverse events or new neurological deficits attributed to 5-AZA infusions. All five patients with ependymoma who received 5-AZA infusions had progressive disease. Two of the five patients, however, were noted to have decrease in the size of at least one intraventricular lesion.Conclusion5-AZA can be infused into the fourth ventricle or posterior fossa tumor resection cavity without causing neurological toxicity. Future studies with higher doses and/or increased dosing frequency are warranted.

Intracranial aneurysms (IA) are local dilatations in cerebral arteries that predominantly affect the circle of Willis. Occurring in approximately 2–5% of adults, these weakened areas are susceptible to rupture, leading to subarachnoid hemorrhage (SAH), a type of hemorrhagic stroke. Due to its early age of onset and poor prognosis, SAH accounts for > 25% of years lost for all stroke victims under the age of 65. In this review, we describe the cerebrovascular pathology associated with intracranial aneurysms. To understand IA genetics, we summarize syndromes with elevated incidence, genome-wide association studies (GWAS), whole exome studies on IA-affected families, and recent research that established definitive roles for Thsd1 (Thrombospondin Type 1 Domain Containing Protein 1) and Sox17 (SRY-box 17) in IA using genetically engineered mouse models. Lastly, we discuss the underlying molecular mechanisms of IA, including defects in vascular endothelial and smooth muscle cells caused by dysfunction in mechanotransduction, Thsd1/FAK (Focal Adhesion Kinase) signaling, and the Transforming Growth Factor β (TGF-β) pathway. As illustrated by THSD1 research, cell adhesion may play a significant role in IA.

Protein tagging is a powerful tool for performing comprehensive analyses of the biological functions of a protein of interest owing to the existence of a wide variety of tags. It becomes indispensable in some cases, such as in tracking protein dynamics in a live cell or adding a peptide epitope due to the lack of optimal antibodies. However, efficiently integrating an array of tags into the gene of interest remains a challenge. Traditional DNA recombinant technology based on type II restriction endonucleases renders protein tagging tedious and inefficient as well as the introduction of an unwanted junction sequence. In our attempt to tag Thrombospondin type 1 domain-containing 1 (THSD1) that we identified as the first intracranial aneurysm gene (Santiago-Sim et al., 2016), we developed a novel precision tagging technique by combinational use of type II and IIS restriction endonucleases (Xu et al., 2017), which generates a seamless clone with high efficiency. Here, we describe a protocol that not only provides a generalized strategy for any gene of interest but also takes its application of 11 different tags in THSD1 as a step-by-step example.

Currently, there is an incomplete understanding of the molecular pathogenesis of meningiomas, the most common primary brain tumor. Several familial syndromes are characterized by increased meningioma risk, and the genetics of these syndromes provides mechanistic insight into sporadic disease. The best defined of these syndromes is neurofibromatosis type 2, which is caused by a mutation in the NF2 gene and has a meningioma incidence of approximately 50%. This finding led to the subsequent discovery that NF2 loss-of-function occurs in up to 60% of sporadic tumors. Other important familial diseases with increased meningioma risk include nevoid basal cell carcinoma syndrome, multiple endocrine neoplasia 1 (MEN1), Cowden syndrome, Werner syndrome, BAP1 tumor predisposition syndrome, Rubinstein-Taybi syndrome, and familial meningiomatosis caused by germline mutations in the SMARCB1 and SMARCE1 genes. For each of these syndromes, the diagnostic criteria, incidence in the population, and frequency of meningioma are presented to review the relevant clinical information for these conditions. The genetic mutations, molecular pathway derangements, and relationship to sporadic disease for each syndrome are described in detail to identify targets for further investigation. Familial syndromes characterized by meningiomas often affect genes and pathways that are also implicated in a subset of sporadic cases, suggesting key molecular targets for therapeutic intervention. Further studies are needed to resolve the functional relevance of specific genes whose significance in sporadic disease remains to be elucidated.

Emerging evidence implicates a wide range of post-transcriptional RNA modifications that play crucial roles in fundamental biological processes including regulating gene expression. Collectively, they are known as epitranscriptomics. Recent studies implicate 3' RNA uridylation, the non-templated addition of uridine(s) to the terminal end of RNA, as a key player in epitranscriptomics. In this review, we describe the functional roles and significance of 3' terminal RNA uridylation that has diverse functions in regulating both mRNAs and non-coding RNAs. In mammals, three Terminal Uridylyl Transferases (TUTases) are primarily responsible for 3' RNA uridylation. These enzymes are also referred to as polyU polymerases. TUTase 1 (TUT1) is implicated in U6 snRNA maturation via uridylation. The TUTases TUT4 and/or TUT7 are the predominant mediators of all other cellular uridylation. Terminal uridylation promotes turnover for many polyadenylated mRNAs, replication-dependent histone mRNAs that lack polyA-tails, and aberrant structured noncoding RNAs. In addition, uridylation regulates biogenesis of a subset of microRNAs and generates isomiRs, sequent variant microRNAs that have altered function in specific cases. For example, the RNA binding protein and proto-oncogene LIN28A and TUT4 work together to polyuridylate pre-let-7, thereby blocking biogenesis and function of the tumor suppressor let-7 microRNA family. In contrast, monouridylation of Group II pre-miRNAs creates an optimal 3' overhang that promotes recognition and subsequent cleavage by the Dicer-TRBP complex that then yields the mature microRNA. Also, uridylation may play a role in non-canonical microRNA biogenesis. The overall significance of 3' RNA uridylation is discussed with an emphasis on mammalian development, gene regulation, and disease, including cancer and Perlman syndrome. We also introduce recent changes to the HUGO-approved gene names for multiple terminal nucleotidyl transferases that affects in part TUTase nomenclature (TUT1/TENT1, TENT2/PAPD4/GLD2, TUT4/ZCCHC11/TENT3A, TUT7/ZCCHC6/TENT3B, TENT4A/PAPD7, TENT4B/PAPD5, TENT5A/FAM46A, TENT5B/FAM46B, TENT5C/FAM46C, TENT5D/FAM46D, MTPAP/TENT6/PAPD1).

Abstract Currently, there is an incomplete understanding of the molecular pathogenesis of meningiomas, the most common primary brain tumor. Several familial syndromes are characterized by increased meningioma risk, and the genetics of these syndromes provides mechanistic insight into sporadic disease. The best defined of these syndromes is neurofibromatosis type 2, which is caused by a mutation in the NF2 gene and has a meningioma incidence of approximately 50%. This finding led to the subsequent discovery that NF2 loss-of-function occurs in up to 60% of sporadic tumors. Other important familial diseases with increased meningioma risk include nevoid basal cell carcinoma syndrome, multiple endocrine neoplasia 1 (MEN1), Cowden syndrome, Werner syndrome, BAP1 tumor predisposition syndrome, Rubinstein-Taybi syndrome, and familial meningiomatosis caused by germline mutations in the SMARCB1 and SMARCE1 genes. For each of these syndromes, the diagnostic criteria, incidence in the population, and frequency of meningioma are presented to review the relevant clinical information for these conditions. The genetic mutations, molecular pathway derangements, and relationship to sporadic disease for each syndrome are described in detail to identify targets for further investigation. Familial syndromes characterized by meningiomas often affect genes and pathways that are also implicated in a subset of sporadic cases, suggesting key molecular targets for therapeutic intervention. Further studies are needed to resolve the functional relevance of specific genes whose significance in sporadic disease remains to be elucidated.

BACKGROUND & AIMSCachexia, which includes muscle wasting, is a frequent complication of pancreatic cancer. There are no therapies that reduce cachexia and increase patient survival, so it is important to learn more about its mechanisms. The zinc transporter ZIP4 promotes growth and metastasis of pancreatic tumors. We investigated its effects on muscle catabolism via extracellular vesicle (EV)-mediated stimulation of mitogen-activated protein kinase 14 (p38 MAPK).METHODSWe studied nude mice with orthotopic tumors grown from human pancreatic cancer cell lines (AsPC-1 and BxPC-3); tumors were removed 8 days after cell injection and analyzed by histology. Mouse survival was analyzed by Kaplan-Meier curves. ZIP4 was knocked down in AsPC-1 and BxPC-3 cells with small hairpin RNAs; cells with empty vectors were used as controls. Muscle tissues were collected from mice and analyzed by histology and immunohistochemistry. Conditioned media from cell lines and 3-dimensional spheroid/organoid cultures of cancer cells were applied to C2C12 myotubes. The myotubes and the media were analyzed by immunoblots, enzyme-linked immunosorbent assays, and immunofluorescence microscopy. EVs were isolated from conditioned media and analyzed by immunoblots.RESULTSMice with orthotopic tumors grown from pancreatic cancer cells with knockdown of ZIP4 survived longer and lost less body weight and muscle mass than mice with control tumors. Conditioned media from cancer cells activated p38 MAPK, induced expression of F-box protein 32 and UBR2 in C2C12 myotubes, and also led to loss of myofibrillar protein myosin heavy chain and myotube thinning. Knockdown of ZIP4 in cancer cells reduced these effects. ZIP4 knockdown also reduced pancreatic cancer cell release of heat shock protein (HSP) 70 and HSP90, which are associated with EVs, by decreasing CREB-regulated expression of RAB27B.CONCLUSIONSZIP4 promotes growth of orthotopic pancreatic tumors in mice and loss of muscle mass by activating CREB-regulated expression of RAB27B, required for release of EVs from pancreatic cancer cells. These EVs activate p38 MAPK and induce expression of F-box protein 32 and UBR2 in myotubes, leading to loss of myofibrillar myosin heavy chain and myotube thinning. Strategies to disrupt these pathways might be developed to reduce pancreatic cancer progression and accompanying cachexia.

Background/Aims: We recently discovered that harmful variants in THSD1 (Thrombospondin type-1 domain-containing protein 1) likely cause intracranial aneurysm and subarachnoid hemorrhage in a subset of both familial and sporadic patients with supporting evidence from two vertebrate models. The current study seeks to elucidate how THSD1 and patient-identified variants function molecularly in focal adhesions. Methods: Co-immunostaining and co-immunoprecipitation were performed to define THSD1 subcellular localization and interacting partners. Transient expression of patient-identified THSD1 protein variants and siRNA-mediated loss-of-function THSD1 were used to interrogate gene function in focal adhesion and cell attachment to collagen I in comparison to controls. Results: THSD1 is a novel nascent adhesion protein that co-localizes with several known markers such as FAK, talin, and vinculin, but not with mature adhesion marker zyxin. Furthermore, THSD1 forms a multimeric protein complex with FAK/talin/vinculin, wherein THSD1 promotes talin binding to FAK but not to vinculin, a key step in nascent adhesion assembly. Accordingly, THSD1 promotes mature adhesion formation and cell attachment, while its rare variants identified in aneurysm patients show compromised ability. Interestingly, THSD1 also localizes at different stages of endosomes. Clathrin-mediated but not caveolae-mediated endocytosis pathway is involved in THSD1 intracellular trafficking, which positively regulates THSD1-induced focal adhesion assembly, in contrast to the traditional role of endosomes in termination of integrin signals. Conclusions: The data suggest that THSD1 functions at the interface between endosome dynamics and nascent focal adhesion assembly that is impaired by THSD1 rare variants identified from intracranial aneurysm patients.