Loss of an H3K9me anchor rescues laminopathy-linked changes in nuclear organization and muscle function in an Emery-Dreifuss muscular dystrophy model.

Mutations in the nuclear structural protein lamin A produce rare, tissue-specific diseases called laminopathies. The introduction of a human Emery-Dreifuss muscular dystrophy (EDMD)-inducing mutation into the C. elegans lamin (LMN-Y59C), recapitulates many muscular dystrophy phenotypes, and correlates with hyper-sequestration of a heterochromatic array at the nuclear periphery in muscle cells. Using muscle-specific emerin Dam-ID in worms, we monitored the effects of the mutation on endogenous chromatin. An increased contact with the nuclear periphery along chromosome arms, and an enhanced release of chromosomal centers, coincided with the disease phenotypes of reduced locomotion and compromised sarcomere integrity. The coupling of the LMN-Y59C mutation with the ablation of CEC-4, a chromodomain protein that anchors H3K9-methylated chromatin at the nuclear envelope (NE), suppressed the muscle-associated disease phenotypes. Deletion of cec-4 also rescued LMN-Y59C-linked alterations in chromatin organization and some changes in transcription. Sequences that changed position in the LMN-Y59C mutant, are enriched for E2F (EFL-2)-binding sites, consistent with previous studies suggesting that altered Rb-E2F interaction with lamin A may contribute to muscle dysfunction. In summary, we were able to counteract the dominant muscle-specific defects provoked by LMNA mutation by the ablation of a lamin-associated H3K9me anchor, suggesting a novel therapeutic pathway for EDMD.

Llgl1 mediates timely epicardial emergence and establishment of an apical laminin sheath around the trabeculating cardiac ventricle.

During heart development, the embryonic ventricle becomes enveloped by the epicardium, which adheres to the outer apical surface of the heart. This is concomitant with onset of ventricular trabeculation, where a subset of cardiomyocytes lose apicobasal polarity and delaminate basally from the ventricular wall. Llgl1 regulates the formation of apical cell junctions and apicobasal polarity, and we investigated its role in ventricular wall maturation. We found that llgl1 mutant zebrafish embryos exhibit aberrant apical extrusion of ventricular cardiomyocytes. While investigating apical cardiomyocyte extrusion, we identified a basal-to-apical shift in laminin deposition from the internal to the external ventricular wall. We find that epicardial cells express several laminin subunits as they adhere to the ventricle, and that the epicardium is required for laminin deposition on the ventricular surface. In llgl1 mutants, timely establishment of the epicardial layer is disrupted due to delayed emergence of epicardial cells, resulting in delayed apical deposition of laminin on the ventricular surface. Together, our analyses reveal an unexpected role for Llgl1 in correct timing of epicardial development, supporting integrity of the ventricular myocardial wall.

Functional diversity of laminins.

Laminins are a large family of conserved, multidomain trimeric basement membrane proteins that contribute to the structure of extracellular matrix and influence the behavior of associated cells, such as adhesion, differentiation, migration, phenotype stability, and resistance to anoikis. In lower organisms such as Hydra there is only one isoform of laminin, but higher organisms have at least 16 trimeric isoforms with varying degrees of cell/tissue specificity. In vitro protein and cell culture studies, gene manipulation in animals, and laminin gene mutations in human diseases have provided insight into the specific functions of some laminins, but the biological roles of many isoforms are still largely unexplored, mainly owing to difficulties in isolating them in pure form from tissues or cells. In this review, we elucidate the evolution of laminins, describe their molecular complexity, and explore the current knowledge of their diversity and functional aspects, including laminin-mediated signaling via membrane receptors, in vitro cell biology, and involvement in various tissues gained from animal model and human disease studies. The potential use of laminins in cell biology research and biotechnology is discussed.

Polymerizing laminins in development, health, and disease.

Polymerizing laminins are multi-domain basement membrane (BM) glycoproteins that self-assemble into cell-anchored planar lattices to establish the initial BM scaffold. Nidogens, collagen-IV and proteoglycans then bind to the scaffold at different domain loci to create a mature BM. The LN domains of adjacent laminins bind to each other to form a polymer node, while the LG domains attach to cytoskeletal-anchoring integrins and dystroglycan, as well as to sulfatides and heparan sulfates. The polymer node, the repeating unit of the polymer scaffold, is organized into a near-symmetrical triskelion. The structure, recently solved by cryo-electron microscopy in combination with AlphaFold2 modeling and biochemical studies, reveals how the LN surface residues interact with each other and how mutations cause failures of self-assembly in an emerging group of diseases, the LN-lamininopathies, that include LAMA2-related dystrophy and Pierson syndrome.

Timely Schwann cell division drives peripheral myelination in vivo via the laminin/cAMP pathway.

Schwann cells (SCs) migrate along peripheral axons and divide intensively to generate the right number of cells prior to axonal ensheathment; however, little is known regarding the temporal and molecular control of their division and its impact on myelination. We report that Sil, a spindle pole protein associated with autosomal recessive primary microcephaly, is required for temporal mitotic exit of SCs. In sil-deficient cassiopeia (csp-/-) mutants, SCs fail to radially sort and myelinate peripheral axons. Elevation of cAMP, but not Rac1 activity, in csp-/- restores myelin ensheathment. Most importantly, we show a significant decrease in laminin expression within csp-/- posterior lateral line nerve and that forcing Laminin 2 expression in csp-/- fully restores the ability of SCs to myelinate. Thus, we demonstrate an essential role for timely SC division in mediating laminin expression to orchestrate radial sorting and peripheral myelination in vivo.

Release of miR-29 Target Laminin C2 Improves Skin Repair.

miRNAs are small noncoding RNAs that regulate mRNA targets in a cell-specific manner. miR-29 is expressed in murine and human skin, where it may regulate functions in skin repair. Cutaneous wound healing model in miR-29a/b1 gene knockout mice was used to identify miR-29 targets in the wound matrix, where angiogenesis and maturation of provisional granulation tissue was enhanced in response to genetic deletion of miR-29. Consistently, antisense-mediated inhibition of miR-29 promoted angiogenesis in vitro by autocrine and paracrine mechanisms. These processes are likely mediated by miR-29 target mRNAs released upon removal of miR-29 to improve cell-matrix adhesion. One of these, laminin (Lam)-c2 (also known as laminin γ2), was strongly up-regulated during skin repair in the wound matrix of knockout mice. Unexpectedly, Lamc2 was deposited in the basal membrane of endothelial cells in blood vessels forming in the granulation tissue of knockout mice. New blood vessels showed punctate interactions between Lamc2 and integrin α6 (Itga6) along the length of the proto-vessels, suggesting that greater levels of Lamc2 may contribute to the adhesion of endothelial cells, thus assisting angiogenesis within the wound. These findings may be of translational relevance, as LAMC2 was deposited at the leading edge in human wounds, where it formed a basal membrane for endothelial cells and assisted neovascularization. These results suggest a link between LAMC2, improved angiogenesis, and re-epithelialization.

Accelerated maturation of ARPE-19 cells for the translational assessment of gene therapy.

The human retinal pigment epithelium (RPE) cell line ARPE-19 is widely used as an alternative to primary RPE despite losing many features of primary RPE. We aimed to determine whether a combination of RPE-specific laminin (LN) and nicotinamide (NAM) could improve ARPE-19 redifferentiation to resemble mature RPE and improve the assessment of RPE-specific gene therapy strategies. ARPE-19 cells were propagated on tissue culture plastic supplemented with NAM and human recombinant LN521-coating. RPE maturation was performed by immunocytochemistry and gene expression by qPCR. Viral transduction experiments with adeno-associated virus (AAV)1 or AAV2, carrying a VMD2-driven GFP, were assessed at 2- and 4-weeks post-plating in the different culturing conditions with a low multiplicity of infection. The combination of LN521 coating with NAM supplementation promoted cytoskeletal and tight junction protein reorganization. The expression of maturation markers bestrophin-1 and RPE 65 was promoted concomitantly with a reduction of several epithelial-mesenchymal transition markers, such as TNF-α, TGF-ß, CDH2, and vimentin. Redifferentiated ARPE-19 transduced at low multiplicity of infection of both AAV1- and AAV2-VMD2-GFP. Expression of GFP was detected at 2 weeks and increased at 4 weeks post-plating. AAV1 exhibited a greater expression efficacy compared to AAV2 in maturated ARPE-19 cells already after 2 weeks with increased efficiency after 4 weeks. Our study demonstrates an improved maturation protocol for ARPE-19 cells in vitro, mimicking an in vivo phenotype with the expression of signature genes and improved morphology. Viral-mediated RPE-specific gene expression demonstrates that the combination cultures mimic in vivo AAV tropism essential to test new gene therapies for RPE-centered diseases.

LAMA3 Promotes Tumorigenesis of Oral Squamous Cell Carcinoma by METTL3-Mediated N6-Methyladenosine Modification.

Laminin subunit alpha 3 (LAMA3) is a cancer regulator. However, its effects and regulatory pathways in oral squamous cell carcinoma (OSCC) progression remain unknown. This research aimed to determine the influence of LAMA3 regulation via methyltransferase-like 3 (METTL3) on OSCC progression. Using quantitative real-time polymerase chain reaction and bioinformatics analysis, the expression levels of LAMA3 and METTL3 in OSCC tissues were examined. The functional roles of LAMA3 and METTL3 were analyzed using cell functional experiments. Using methylated RNA immunoprecipitation and mRNA stability assays, LAMA3 and METTL3 regulation was investigated. In OSCC tissues, LAMA3 was upregulated. LAMA3 inhibition hampered OSCC cell proliferation, invasion, and migration while its overexpression facilitated OSCC cell progression. METTL3 serves as a crucial upstream regulator of LAMA3 in OSCC and upregulates LAMA3 expression via an m6A-dependent mechanism. The low METTL3 expression partially restored the enhanced malignant phenotype induced by LAMA3 overexpression. Our findings indicate that METTL3 and LAMA3 act as pro-oncogenic factors in OSCC, with METTL3 promoting OSCC malignancy via m6A modification-dependent stabilization of LAMA3 transcripts, representing a novel regulatory mechanism in OSCC.

Ramipril therapy in integrin α1-null, autosomal recessive Alport mice triples lifespan: mechanistic clues from RNA-seq analysis.

The standard of care for patients with Alport syndrome (AS) is angiotensin-converting enzyme (ACE) inhibitors. In autosomal recessive Alport (ARAS) mice, ACE inhibitors double lifespan. We previously showed that deletion of Itga1 in Alport mice [double-knockout (DKO) mice] increased lifespan by 50%. This effect seemed dependent on the prevention of laminin 211-mediated podocyte injury. Here, we treated DKO mice with vehicle or ramipril starting at 4 weeks of age. Proteinuria and glomerular filtration rates were measured at 5-week intervals. Glomeruli were analyzed for laminin 211 deposition in the glomerular basement membrane (GBM) and GBM ultrastructure was analyzed using transmission electron microscopy (TEM). RNA sequencing (RNA-seq) was performed on isolated glomeruli at all time points and the results were compared with cultured podocytes overlaid (or not) with recombinant laminin 211. Glomerular filtration rate declined in ramipril-treated DKO mice between 30 and 35 weeks. Proteinuria followed these same patterns with normalization of foot process architecture in ramipril-treated DKO mice. RNA-seq revealed a decline in the expression of Foxc2, nephrin (Nphs1), and podocin (Nphs2) mRNAs, which was delayed in the ramipril-treated DKO mice. GBM accumulation of laminin 211 was delayed in ramipril-treated DKO mice, likely due to a role for α1ß1 integrin in CDC42 activation in Alport mesangial cells, which is required for mesangial filopodial invasion of the subendothelial spaces of the glomerular capillary loops. Ramipril synergized with Itga1 knockout, tripling lifespan compared with untreated ARAS mice. © 2023 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A mutation-independent approach for muscular dystrophy via upregulation of a modifier gene.

Neuromuscular disorders are often caused by heterogeneous mutations in large, structurally complex genes. Targeting compensatory modifier genes could be beneficial to improve disease phenotypes. Here we report a mutation-independent strategy to upregulate the expression of a disease-modifying gene associated with congenital muscular dystrophy type 1A (MDC1A) using the CRISPR activation system in mice. MDC1A is caused by mutations in LAMA2 that lead to nonfunctional laminin-α2, which compromises the stability of muscle fibres and the myelination of peripheral nerves. Transgenic overexpression of Lama1, which encodes a structurally similar protein called laminin-α1, ameliorates muscle wasting and paralysis in mouse models of MDC1A, demonstrating its importance as a compensatory modifier of the disease1. However, postnatal upregulation of Lama1 is hampered by its large size, which exceeds the packaging capacity of vehicles that are clinically relevant for gene therapy. We modulate expression of Lama1 in the dy2j/dy2j mouse model of MDC1A using an adeno-associated virus (AAV9) carrying a catalytically inactive Cas9 (dCas9), VP64 transactivators and single-guide RNAs that target the Lama1 promoter. When pre-symptomatic mice were treated, Lama1 was upregulated in skeletal muscles and peripheral nerves, which prevented muscle fibrosis and paralysis. However, for many disorders it is important to investigate the therapeutic window and reversibility of symptoms. In muscular dystrophies, it has been hypothesized that fibrotic changes in skeletal muscle are irreversible. However, we show that dystrophic features and disease progression were improved and reversed when the treatment was initiated in symptomatic dy2j/dy2j mice with apparent hindlimb paralysis and muscle fibrosis. Collectively, our data demonstrate the feasibility and therapeutic benefit of CRISPR-dCas9-mediated upregulation of Lama1, which may enable mutation-independent treatment for all patients with MDC1A. This approach has a broad applicability to a variety of disease-modifying genes and could serve as a therapeutic strategy for many inherited and acquired diseases.

Lentiviral expression of wild-type LAMA3A restores cell adhesion in airway basal cells from children with epidermolysis bullosa.

The hallmark of epidermolysis bullosa (EB) is fragile attachment of epithelia due to genetic variants in cell adhesion genes. We describe 16 EB patients treated in the ear, nose, and throat department of a tertiary pediatric hospital linked to the United Kingdom's national EB unit between 1992 and 2023. Patients suffered a high degree of morbidity and mortality from laryngotracheal stenosis. Variants in laminin subunit alpha-3 (LAMA3) were found in 10/15 patients where genotype was available. LAMA3 encodes a subunit of the laminin-332 heterotrimeric extracellular matrix protein complex and is expressed by airway epithelial basal stem cells. We investigated the benefit of restoring wild-type LAMA3 expression in primary EB patient-derived basal cell cultures. EB basal cells demonstrated weak adhesion to cell culture substrates, but could otherwise be expanded similarly to non-EB basal cells. In vitro lentiviral overexpression of LAMA3A in EB basal cells enabled them to differentiate in air-liquid interface cultures, producing cilia with normal ciliary beat frequency. Moreover, transduction restored cell adhesion to levels comparable to a non-EB donor culture. These data provide proof of concept for a combined cell and gene therapy approach to treat airway disease in LAMA3-affected EB.

Genetic blueprint of congenital muscular dystrophies with brain malformations in Egypt: A report of 11 families.

Congenital muscular dystrophies (CMDs) are a group of rare muscle disorders characterized by early onset hypotonia and motor developmental delay associated with brain malformations with or without eye anomalies in the most severe cases. In this study, we aimed to uncover the genetic basis of severe CMD in Egypt and to determine the efficacy of whole exome sequencing (WES)-based genetic diagnosis in this population. We recruited twelve individuals from eleven families with a clinical diagnosis of CMD with brain malformations that fell into two groups: seven patients with suspected dystroglycanopathy and five patients with suspected merosin-deficient CMD. WES was analyzed by variant filtering using multiple approaches including splicing and copy number variant (CNV) analysis. We identified likely pathogenic variants in FKRP in two cases and variants in POMT1, POMK, and B3GALNT2 in three individuals. All individuals with merosin-deficient CMD had truncating variants in LAMA2. Further analysis in one of the two unsolved cases showed a homozygous protein-truncating variant in Feline Leukemia Virus subgroup C Receptor 1 (FLVCR1). FLVCR1 loss of function has never been previously reported. Yet, loss of function of its paralog, FLVCR2, causes lethal hydranencephaly-hydrocephaly syndrome (Fowler Syndrome) which should be considered in the differential diagnosis for dystroglycanopathy. Overall, we reached a diagnostic rate of 86% (6/7) for dystroglycanopathies and 100% (5/5) for merosinopathy. In conclusion, our results provide further evidence that WES is an important diagnostic method in CMD in developing countries to improve the diagnostic rate, management plan, and genetic counseling for these disorders.

Identification of established and novel extracellular matrix components in glioblastoma as targets for angiogenesis and prognosis.

Glioblastomas (GBM) are aggressive tumors known for their heterogeneity, rapid proliferation, treatment resistance, and extensive vasculature. Angiogenesis, the formation of new vessels, involves endothelial cell (EC) migration and proliferation. Various extracellular matrix (ECM) molecules regulate EC survival, migration, and proliferation. Culturing human brain EC (HBMEC) on GBM-derived ECM revealed a decrease in EC numbers compared to controls. Through in silico analysis, we explored ECM gene expression differences between GBM and brain normal glia cells and the impact of GBM microenvironment on EC ECM transcripts. ECM molecules such as collagen alpha chains (COL4A1, COL4A2, p < 0.0001); laminin alpha (LAMA4), beta (LAMB2), and gamma (LAMC1) chains (p < 0.0005); neurocan (NCAN), brevican (BCAN) and versican (VCAN) (p < 0.0005); hyaluronan synthase (HAS) 2 and metalloprotease (MMP) 2 (p < 0.005); MMP inhibitors (TIMP1-4, p < 0.0005), transforming growth factor beta-1 (TGFB1) and integrin alpha (ITGA3/5) (p < 0.05) and beta (ITGB1, p < 0.0005) chains showed increased expression in GBM. Additionally, GBM-influenced EC exhibited elevated expression of COL5A3, COL6A1, COL22A1 and COL27A1 (p < 0.01); LAMA1, LAMB1 (p < 0.001); fibulins (FBLN1/2, p < 0.01); MMP9, HAS1, ITGA3, TGFB1, and wingless-related integration site 9B (WNT9B) (p < 0.01) compared to normal EC. Some of these molecules: COL5A1/3, COL6A1, COL22/27A1, FBLN1/2, ITGA3/5, ITGB1 and LAMA1/B1 (p < 0.01); NCAN, HAS1, MMP2/9, TIMP1/2 and TGFB1 (p < 0.05) correlated with GBM patient survival. In conclusion, this study identified both established and novel ECM molecules regulating GBM angiogenesis, suggesting NCAN and COL27A1 are new potential prognostic biomarkers for GBM.

Lysosomes and the pathogenesis of merosin-deficient congenital muscular dystrophy.

Congenital muscular dystrophy type 1A (MDC1A), the most common congenital muscular dystrophy in Western countries, is caused by recessive mutations in LAMA2, the gene encoding laminin alpha 2. Currently, no cure or disease modifying therapy has been successfully developed for MDC1A. Examination of patient muscle biopsies revealed altered distribution of lysosomes. We hypothesized that this redistribution was a novel and potentially druggable aspect of disease pathogenesis. We explored this hypothesis using candyfloss (caf), a zebrafish model of MDC1A. We found that lysosome distribution in caf zebrafish was also abnormal. This altered localization was significantly associated with fiber detachment and could be prevented by blocking myofiber detachment. Overexpression of transcription factor EB, a transcription factor that promotes lysosomal biogenesis, led to increased lysosome content and decreased fiber detachment. We conclude that genetic manipulation of the lysosomal compartment is able to alter the caf zebrafish disease process, suggesting that lysosome function may be a target for disease modification.

Sarcospan increases laminin-binding capacity of α-dystroglycan to ameliorate DMD independent of Galgt2.

In Duchenne muscular dystrophy (DMD), mutations in dystrophin result in a loss of the dystrophin-glycoprotein complex (DGC) at the myofiber membrane, which functions to connect the extracellular matrix with the intracellular actin cytoskeleton. The dystroglycan subcomplex interacts with dystrophin and spans the sarcolemma where its extensive carbohydrates (matriglycan and CT2 glycan) directly interact with the extracellular matrix. In the current manuscript, we show that sarcospan overexpression enhances the laminin-binding capacity of dystroglycan in DMD muscle by increasing matriglycan glycosylation of α-dystroglycan. Furthermore, we find that this modification is not affected by loss of Galgt2, a glycotransferase, which catalyzes the CT2 glycan. Our findings reveal that the matriglycan carbohydrates, and not the CT2 glycan, are necessary for sarcospan-mediated amelioration of DMD. Overexpression of Galgt2 in the DMD mdx murine model prevents muscle pathology by increasing CT2 modified α-dystroglycan. Galgt2 also increases expression of utrophin, which compensates for the loss of dystrophin in DMD muscle. We found that combined loss of Galgt2 and dystrophin reduced utrophin expression; however, it did not interfere with sarcospan rescue of disease. These data reveal a partial dependence of sarcospan on Galgt2 for utrophin upregulation. In addition, sarcospan alters the cross-talk between the adhesion complexes by decreasing the association of integrin ß1D with dystroglycan complexes. In conclusion, sarcospan functions to re-wire the cell to matrix connections by strengthening the cellular adhesion and signaling, which, in turn, increases the resilience of the myofiber membrane.

Extracellular matrix marker LAMC2 targets ZEB1 to promote TNBC malignancy via up-regulating CD44/STAT3 signaling pathway.

BACKGROUND: Triple negative breast cancer (TNBC) is a heterogeneous and aggressive disease characterized by a high risk of mortality and poor prognosis. It has been reported that Laminin γ2 (LAMC2) is highly expressed in a variety of tumors, and its high expression is correlated with cancer development and progression. However, the function and mechanism by which LAMC2 influences TNBC remain unclear. METHODS: Kaplan-Meier survival analysis and Immunohistochemical (IHC) staining were used to examine the expression level of LAMC2 in TNBC. Subsequently, cell viability assay, wound healing and transwell assay were performed to detect the function of LAMC2 in cell proliferation and migration. A xenograft mouse model was used to assess tumorigenic function of LAMC2 in vivo. Luciferase reporter assay and western blot were performed to unravel the underlying mechanism. RESULTS: In this study, we found that higher expression of LAMC2 significantly correlated with poor survival in the TNBC cohort. Functional characterization showed that LAMC2 promoted cell proliferation and migration capacity of TNBC cell lines via up-regulating CD44. Moreover, LAMC2 exerted oncogenic roles in TNBC through modulating the expression of epithelial-mesenchymal transition (EMT) markers. Luciferase reporter assay verified that LAMC2 targeted ZEB1 to promote its transcription. Interestingly, LAMC2 regulated cell migration in TNBC via STAT3 signaling pathway. CONCLUSION: LAMC2 targeted ZEB1 via activating CD44/STAT3 signaling pathway to promote TNBC proliferation and migration, suggesting that LAMC2 could be a potential therapeutic target in TNBC patients.

Laminin alpha 5 regulates mammary gland remodeling through luminal cell differentiation and Wnt4-mediated epithelial crosstalk.

Epithelial attachment to the basement membrane (BM) is essential for mammary gland development, yet the exact roles of specific BM components remain unclear. Here, we show that Laminin α5 (Lama5) expression specifically in the luminal epithelial cells is necessary for normal mammary gland growth during puberty, and for alveologenesis during pregnancy. Lama5 loss in the keratin 8-expressing cells results in reduced frequency and differentiation of hormone receptor expressing (HR+) luminal cells. Consequently, Wnt4-mediated crosstalk between HR+ luminal cells and basal epithelial cells is compromised during gland remodeling, and results in defective epithelial growth. The effects of Lama5 deletion on gland growth and branching can be rescued by Wnt4 supplementation in the in vitro model of branching morphogenesis. Our results reveal a surprising role for BM-protein expression in the luminal mammary epithelial cells, and highlight the function of Lama5 in mammary gland remodeling and luminal differentiation.

HIF-1α-mediated LAMC1 overexpression is an unfavorable predictor of prognosis for glioma patients: evidence from pan-cancer analysis and validation experiments.

BACKGROUND: Laminin subunit gamma-1 (LAMC1) is a major extracellular matrix molecule involved in the tumor microenvironment. Knowledge of the biological features and clinical relevance of LAMC1 in cancers remains limited. METHODS: We conducted comprehensive bioinformatics analysis of LAMC1 gene expression and clinical relevance in pan-cancer datasets of public databases and validated LAMC1 expression in glioma tissues and cell lines. The association and regulatory mechanism between hypoxia inducible factor-1α (HIF-1α) and LAMC1 expression were explored. RESULTS: LAMC1 expression in most cancers in The Cancer Genome Atlas (TCGA) including glioma was significantly higher than that in normal tissues, which had a poor prognosis and were related to various clinicopathological features. Data from the Chinese Glioma Genome Atlas also showed high expression of LAMC1 in glioma associated with poor prognoses. In clinical glioma tissues, LAMC1 protein was highly expressed and correlated to poor overall survival. LAMC1 knockdown in Hs683 glioma cells attenuated cell proliferation, migration, and invasion, while overexpression of LAMC1 in U251 cells leads to the opposite trend. Most TCGA solid cancers including glioma showed enhancement of HIF-1α expression. High HIF-1α expression leads to adverse prognosis in gliomas, besides, HIF-1α expression was positively related to LAMC1. Mechanistically, HIF-1α directly upregulated LAMC1 promotor activity. Hypoxia (2% O2)-treated Hs683 and U251 cells exhibited upregulated HIF-1α and LAMC1 expression, which was significantly attenuated by HIF-1α inhibitor YC-1 and accompanied by attenuated cell proliferation and invasion. CONCLUSIONS: High expression of LAMC1 in some solid tumors including gliomas suggests a poor prognosis. The hypoxic microenvironment in gliomas activates the HIF-1α/LAMC1 signaling, thereby promoting tumor progression. Targeted intervention on the HIF-1α/LAMC1 signaling attenuates cell growth and invasion, suggesting a new strategy for glioma treatment.

Genetic profile of Brazilian patients with LAMA2-related dystrophies.

LAMA2-related dystrophies (LAMA2-RD) constitute a rare neuromuscular disorder with a broad spectrum of phenotypic severity. Our understanding of the genotype-phenotype correlations in this condition remains incomplete, and reliable clinical data for clinical trial readiness is limited. In this retrospective study, we reviewed the genetic data and medical records of 114 LAMA2-RD patients enrolled at seven research centers in Brazil. We identified 58 different pathogenic variants, including 21 novel ones. Six variants were more prevalent and were present in 81.5% of the patients. Notably, the c.1255del, c.2049_2050del, c.3976 C>T, c.5234+1G>A, and c.4739dup variants were found in patients unable to walk and without cortical malformation. In contrast, the c.2461A>C variant was present in patients who could walk unassisted. Among ambulatory patients, missense variants were more prevalent (p < 0.0001). Although no specific hotspot regions existed in the LAMA2, 51% of point mutations were in the LN domain, and 88% of the missense variants were found within this domain. Functional analysis was performed in one intronic variant (c.4960-17C>A) and revealed an out-of-frame transcript, indicating that the variant creates a cryptic splicing site (AG). Our study has shed light on crucial phenotype-genotype correlations and provided valuable insights, particularly regarding the Latin American population.

Critical role of Lama4 for hematopoiesis regeneration and acute myeloid leukemia progression.

Impairment of normal hematopoiesis and leukemia progression are 2 well-linked processes during leukemia development and are controlled by the bone marrow (BM) niche. Extracellular matrix proteins, including laminin, are important BM niche components. However, their role in hematopoiesis regeneration and leukemia is unknown. Laminin α4 (Lama4), a major receptor-binding chain of several laminins, is altered in BM niches in mice with acute myeloid leukemia (AML). So far, the impact of Lama4 on leukemia progression remains unknown. We here report that Lama4 deletion in mice resulted in impaired hematopoiesis regeneration following irradiation-induced stress, which is accompanied by altered BM niche composition and inflammation. Importantly, in a transplantation-induced MLL-AF9 AML mouse model, we demonstrate accelerated AML progression and relapse in Lama4-/- mice. Upon AML exposure, Lama4-/- mesenchymal stem cells (MSCs) exhibited dramatic molecular alterations, including upregulation of inflammatory cytokines that favor AML growth. Lama4-/- MSCs displayed increased antioxidant activities and promoted AML stem cell proliferation and chemoresistance to cytarabine, which was accompanied by increased mitochondrial transfer from the MSCs to AML cells and reduced reactive oxygen species in AML cells in vitro. Similarly, we detected lower levels of reactive oxygen species in AML cells from Lama4-/- mice post-cytarabine treatment. Notably, LAMA4 inhibition or knockdown in human MSCs promoted human AML cell proliferation and chemoprotection. Together, our study for the first time demonstrates the critical role of Lama4 in impeding AML progression and chemoresistance. Targeting Lama4 signaling pathways may offer potential new therapeutic options for AML.