LncRNA FOXD2-AS1 promotes the growth, invasion and migration of OSCC cells by regulating the MiR-185-5p/PLOD1/Akt/mTOR pathway.

Although lncRNAs are recognized to contribute to the development of oral squamous-cell carcinoma (OSCC), their exact function in invasion and cell migration is not clear. In this research, we explored the molecular and cellular mechanisms of FOXD2-AS1 in OSCC. Prognostic and bioinformatics analyses were used to test for the differential expression of FOXD2-AS1-PLOD1. Following FOXD2-AS1 suppression or overexpression, changes in cell viability were measured using the CCK-8 test; changes in cell migration and invasion abilities were measured using the migration and the Transwell assay. The expression of associated genes and proteins was found using Western blot and RT-qPCR. Analysis of luciferase reporter genes was done to look for regulatory connections between various molecules. The FOXD2-AS1-PLOD1 pair, which was highly expressed in OSCC, was analyzed and experimentally verified to be closely related to the prognosis of OSCC, and a nomogram model and correction curve were constructed. The inhibition of FOXD2-AS1 resulted in the reduction of cell activity, migration, invasion ability and changes in genes related to invasion and migration. In vivo validation showed that inhibition of FOXD2-AS1 expression slowed tumor growth, and related proteins changed accordingly. The experiments verified that FOXD2-AS1 negatively regulated miR-185-5 p and that miR-185-5 p negatively regulated PLOD1. In addition, it was found that the expression of PLOD1, p-Akt and p-mTOR proteins in OSCC cells was reduced by the inhibition of FOXD2-AS1, and FOXD2-AS1 and PLOD1 were closely related to the Akt/mTOR pathway. Increased expression of FOXD2-AS1 promotes OSCC growth, invasion and migration, which is important in part by targeting miR-185-5 p/PLOD1/Akt/mTOR pathway activity.

PLOD2, a key factor for MRL MSC metabolism and chondroprotective properties.

BACKGROUND: Initially discovered for its ability to regenerate ear holes, the Murphy Roth Large (MRL) mouse has been the subject of multiple research studies aimed at evaluating its ability to regenerate other body tissues and at deciphering the mechanisms underlying it. These enhanced abilities to regenerate, retained during adulthood, protect the MRL mouse from degenerative diseases such as osteoarthritis (OA). Here, we hypothesized that mesenchymal stromal/stem cells (MSC) derived from the regenerative MRL mouse could be involved in their regenerative potential through the release of pro-regenerative mediators. METHOD: To address this hypothesis, we compared the secretome of MRL and BL6 MSC and identified several candidate molecules expressed at significantly higher levels by MRL MSC than by BL6 MSC. We selected one candidate, Plod2, and performed functional in vitro assays to evaluate its role on MRL MSC properties including metabolic profile, migration, and chondroprotective effects. To assess its contribution to MRL protection against OA, we used an experimental model for osteoarthritis induced by collagenase (CiOA). RESULTS: Among the candidate molecules highly expressed by MRL MSC, we focused our attention on procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 (PLOD2). Plod2 silencing induced a decrease in the glycolytic function of MRL MSC, resulting in the alteration of their migratory and chondroprotective abilities in vitro. In vivo, we showed that Plod2 silencing in MRL MSC significantly impaired their capacity to protect mouse from developing OA. CONCLUSION: Our results demonstrate that the chondroprotective and therapeutic properties of MRL MSC in the CiOA experimental model are in part mediated by PLOD2.

Spindle cell rhabdomyosarcomas: With TFCP2 rearrangements, and novel EWSR1::ZBTB41 and PLOD2::RBM6 gene fusions. A study of five cases and review of the literature.

AIMS: Spindle-cell/sclerosing rhabdomyosarcomas (SS-RMS) are clinically and genetically heterogeneous. They include three well-defined molecular subtypes, of which those with EWSR1/FUS::TFCP2 rearrangements were described only recently. This study aimed to evaluate five new cases of SS-RMS and to perform a clinicopathological and statistical analysis of all TFCP2-rearranged SS-RMS described in the English literature to more comprehensively characterize this rare tumour type. METHODS AND RESULTS: Cases were retrospectively selected and studied by immunohistochemistry, fluorescence in situ hybridization with EWSR1/FUS and TFCP2 break-apart probes, next-generation sequencing (Archer FusionPlex Sarcoma kit and TruSight RNA Pan-Cancer Panel). The PubMed database was searched for relevant peer-reviewed English reports. Five cases of SS-RMS were found. Three cases were TFCP2 rearranged SS-RMS, having FUSex6::TFCP2ex2 gene fusion in two cases and triple gene fusion EWSR1ex5::TFCP2ex2, VAX2ex2::ALKex2 and VAX2intron2::ALKex2 in one case. Two cases showed rhabdomyoblastic differentiation and spindle-round cell/sclerosing morphology, but were characterized by novel genetic fusions including EWSR1ex8::ZBTB41ex7 and PLOD2ex8::RBM6ex7, respectively. In the statistical analysis of all published cases, CDKN2A or ALK alterations, the use of standard chemotherapy and age at presentation in the range of 18-24 years were negatively correlated to overall survival. CONCLUSION: EWSR1/FUS::TFCP2-rearranged SS-RMS is a rare rhabdomyosarcoma subtype, affecting predominantly young adults with average age at presentation 34 years (median 29.5 years; age range 7-86 years), with a predilection for craniofacial bones, rapid clinical course with frequent bone and lung metastases, and poor prognosis (3-year overall survival rate 28%).

Hypoxia-induced PLOD2 promotes clear cell renal cell carcinoma progression via modulating EGFR-dependent AKT pathway activation.

Clear cell renal cell carcinoma (ccRCC) is a type of kidney cancer that is both common and aggressive, with a rising incidence in recent decades. Hypoxia is a key factor that plays a vital role in the tumorigenesis and metastasis of malignancy. However, the precise mechanisms of hypoxia driving ccRCC progression were not totally uncovered. Our study found that hypoxia level was elevated in ccRCC and might be an independent risk factor of prognosis in ccRCC patients. We identified a key protein PLOD2 was induced under hypoxic conditions and strongly associated with poor prognosis in ccRCC patients. When PLOD2 was depleted, the proliferation and migration of ccRCC cells were reduced in vitro and in vivo, while overexpression of PLOD2 had the opposite effect. Mechanically, the study further revealed that PLOD2 was transcriptionally activated by HIF1A, which binds to a specific promoter region of the PLOD2 gene. PLOD2 was also shown to interact with EGFR, leading to the phosphorylation of the receptor. Furthermore, PLOD2 was responsible for binding to the extracellular domain of EGFR, which ultimately activated the AKT signaling pathway, thus promoting the malignant progression of ccRCC. Treatment with the PLOD2 inhibitor Minoxidil significantly suppressed ccRCC progression by inactivating the EGFR/AKT signaling axis. In summary, the findings of this study shed light on the molecular mechanisms behind PLOD2 expression in ccRCC and suggest that it may serve as a potential predictor and therapeutic target for the clinical prognosis and treatment of ccRCC.

Regulation of EBNA1 protein stability and DNA replication activity by PLOD1 lysine hydroxylase.

Epstein-Barr virus (EBV) is a ubiquitous human γ-herpesvirus that is causally associated with various malignancies and autoimmune disease. Epstein-Barr Nuclear Antigen 1 (EBNA1) is the viral-encoded DNA binding protein required for viral episome maintenance and DNA replication during latent infection in proliferating cells. EBNA1 is known to be a highly stable protein, but the mechanisms regulating protein stability and how this may be linked to EBNA1 function is not fully understood. Proteomic analysis of EBNA1 revealed interaction with Procollagen Lysine-2 Oxoglutarate 5 Dioxygenase (PLOD) family of proteins. Depletion of PLOD1 by shRNA or inhibition with small molecule inhibitors 2,-2' dipyridyl resulted in the loss of EBNA1 protein levels, along with a selective growth inhibition of EBV-positive lymphoid cells. PLOD1 depletion also caused a loss of EBV episomes from latently infected cells and inhibited oriP-dependent DNA replication. Mass spectrometry identified EBNA1 peptides with lysine hydroxylation at K460 or K461. Mutation of K460, but not K461 abrogates EBNA1-driven DNA replication of oriP, but did not significantly affect EBNA1 DNA binding. Mutations in both K460 and K461 perturbed interactions with PLOD1, as well as decreased EBNA1 protein stability. These findings suggest that PLOD1 is a novel interaction partner of EBNA1 that regulates EBNA1 protein stability and function in viral plasmid replication, episome maintenance and host cell survival.

PLOD2 promotes colorectal cancer progression by stabilizing USP15 to activate the AKT/mTOR signaling pathway.

Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) has been reported as an oncogenic gene, affecting various malignant tumors, including endometrial carcinoma, osteosarcoma, and gastric cancer. These effects are mostly due to the enhanced deposition of collagen precursors. However, more studies need to be conducted on how its lysyl hydroxylase function affects cancers like colorectal carcinoma (CRC). Our present results showed that PLOD2 expression was elevated in CRC, and its higher expression was associated with poorer survival. Overexpression of PLOD2 also facilitated CRC proliferation, invasion, and metastasis in vitro and in vivo. In addition, PLOD2 interacted with USP15 by stabilizing it in the cytoplasm and then activated the phosphorylation of AKT/mTOR, thereby promoting CRC progression. Meanwhile, minoxidil was demonstrated to downregulate the expression of PLOD2 and suppress USP15, and the phosphorylation of AKT/mTOR. Our study reveals that PLOD2 plays an oncogenic role in colorectal carcinoma, upregulating USP15 and subsequently activating the AKT/mTOR pathway.

The PLOD2/succinate axis regulates the epithelial-mesenchymal plasticity and cancer cell stemness.

Aberrant accumulation of succinate has been detected in many cancers. However, the cellular function and regulation of succinate in cancer progression is not completely understood. Using stable isotope-resolved metabolomics analysis, we showed that the epithelial mesenchymal transition (EMT) was associated with profound changes in metabolites, including elevation of cytoplasmic succinate levels. The treatment with cell-permeable succinate induced mesenchymal phenotypes in mammary epithelial cells and enhanced cancer cell stemness. Chromatin immunoprecipitation and sequence analysis showed that elevated cytoplasmic succinate levels were sufficient to reduce global 5-hydroxymethylcytosinene (5hmC) accumulation and induce transcriptional repression of EMT-related genes. We showed that expression of procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 (PLOD2) was associated with elevation of cytoplasmic succinate during the EMT process. Silencing of PLOD2 expression in breast cancer cells reduced succinate levels and inhibited cancer cell mesenchymal phenotypes and stemness, which was accompanied by elevated 5hmC levels in chromatin. Importantly, exogenous succinate rescued cancer cell stemness and 5hmC levels in PLOD2-silenced cells, suggesting that PLOD2 promotes cancer progression at least partially through succinate. These results reveal the previously unidentified function of succinate in enhancing cancer cell plasticity and stemness.

Lysyl hydroxylase LH1 promotes confined migration and metastasis of cancer cells by stabilizing Septin2 to enhance actin network.

BACKGROUND: Excessive extracellular matrix deposition and increased stiffness are typical features of solid tumors such as hepatocellular carcinoma (HCC) and pancreatic ductal adenocarcinoma (PDAC). These conditions create confined spaces for tumor cell migration and metastasis. The regulatory mechanism of confined migration remains unclear. METHODS: LC-MS was applied to determine the differentially expressed proteins between HCC tissues and corresponding adjacent tissue. Collective migration and single cell migration microfluidic devices with 6 µm-high confined channels were designed and fabricated to mimic the in vivo confined space. 3D invasion assay was created by Matrigel and Collagen I mixture treat to adherent cells. 3D spheroid formation under various stiffness environment was developed by different substitution percentage GelMA. Immunoprecipitation was performed to pull down the LH1-binding proteins, which were identified by LC-MS. Immunofluorescent staining, FRET, RT-PCR, Western blotting, FRAP, CCK-8, transwell cell migration, wound healing, orthotopic liver injection mouse model and in vivo imaging were used to evaluate the target expression and cellular phenotype. RESULTS: Lysyl hydroxylase 1 (LH1) promoted the confined migration of cancer cells at both collective and single cell levels. In addition, LH1 enhanced cell invasion in a 3D biomimetic model and spheroid formation in stiffer environments. High LH1 expression correlated with poor prognosis of both HCC and PDAC patients, while it also promoted in vivo metastasis. Mechanistically, LH1 bound and stabilized Septin2 (SEPT2) to enhance actin polymerization, depending on the hydroxylase domain. Finally, the subpopulation with high expression of both LH1 and SEPT2 had the poorest prognosis. CONCLUSIONS: LH1 promotes the confined migration and metastasis of cancer cells by stabilizing SEPT2 and thus facilitating actin polymerization.

Apelin promotes osteosarcoma metastasis by upregulating PLOD2 expression via the Hippo signaling pathway and hsa_circ_0000004/miR-1303 axis.

Osteosarcoma is a highly mortal bone tumor, with a high metastatic potential, promoted in part by the enzyme procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2). Increasing level of PLOD2 in osteosarcoma tissue correlates with lymphatic and distant metastasis. The adipokine apelin (APLN) is also found in different cancers and APLN upregulation promotes angiogenesis and metastasis, but its effects on osteosarcoma metastasis are uncertain. We explored APLN functioning in metastatic osteosarcoma. An analysis of records from the Gene Expression Omnibus (GEO) database showed higher levels of APLN expression in osteosarcoma tissue than in normal tissue. Similarly, levels of APLN and PLOD2 mRNA synthesis were upregulated in osteosarcoma tissue. Levels of APLN and PLOD2 protein correlated positively with osteosarcoma clinical stages. APLN increased PLOD2 expression in human osteosarcoma cell lines and cell migration via the mammalian Sterile 20-like kinase 1 (MST1), monopolar spindle-one-binder protein (MOB)1, and YAP cascades, and through hsa_circ_0000004 functioning as a sponge of miR-1303. We also found that knockdown of APLN antagonized lung metastasis in mice with osteosarcoma. APLN may be a therapeutic target in osteosarcoma metastasis.

circRNA PLOD2 promotes tumorigenesis and Warburg effect in colon cancer by the miR-513a-5p/SIX1/LDHA axis.

Increasing evidence has proved that circRNAs might act as potential biomarkers for tumor diagnosis and prognosis. However, the functions and mechanisms of multiple circRNAs in colon cancer remains unclear. Here, we found circPLOD2 was dramatically upregulated in colon cancer tissue and cell lines. In vitro CCK-8, colony formation and transwell assays, and in vivo tumor transplantation assay were performed and explored that circPLOD2 might promote tumor proliferation, migration and invasion in vitro and in vivo. Moreover, based on the analysis of RNA pull-down, RNA immunoprecipitation, luciferase and rescued assays, we confirmed that the interactions between circPLOD2, miR-513a-5p and SIX1. It suggested that circPLOD2 acted as a sponge of miR-513a-5p to regulate the activation of the target gene SIX1. In addition, as a key transcription factor of Warburg effect related genes, SIX1 was proved to enhance the transcriptional expression of LDHA by chromatin immunoprecipitation assay, thereby regulating glycolysis in colon cancer cells. Therefore, we identified that circPLOD2 promoted colon cancer progression through miR-513a-5p/SIX1/LDHA axis, and acted as a new biomarker for colon cancer prognosis and treatment.

Clinicopathological significances of PLOD2, epithelial-mesenchymal transition markers, and cancer stem cells in patients with esophageal squamous cell carcinoma.

BACKGROUND: To examine the expression level of procollagen-lysine2-oxoglutarate 5-dioxygenase 2 (PLOD2) in esophageal squamous cell carcinoma (ESCC) and analyze its correlation with clinicopathological parameters, in order to explore the mechanism of PLOD2 in regulating invasion and metastasis of ESCC. METHODS: Immunohistochemistry was used to detect the expression level of PLOD2 in tumor tissues and paired adjacent tissues of 172 patients with ESCC, and the relationship between PLOD2 expression and clinicopathological parameters was analyzed. The deposition of collagen fibers in tumor was detected by Sirius red staining. The correlation between tumor stem cells and epithelial-mesenchymal transition (EMT) markers ZEB1 was analyzed by multivariate logistic regression. RESULTS: The expression level of PLOD2 in tumor tissues of patients with ESCC (70.35%, 121/172) was significantly higher than that in paired adjacent tissues (29.65%, 51/172; P < .01). The positive expression rate of PLOD2 in ESCC was related to T classification, lymph node metastasis, and pathological tumor node metastasis of a tumor. The expression rates of ZEB1, CD44, and CD133 in ESCC were correlated with T classification, lymph node metastasis and pathological tumor node metastasis. Scarlet red staining showed that collagen fiber deposition in ESCC tissues with high expression of PLOD2 was significantly higher than that in tissues with low expression of PLOD2 (P < .01). A positive correlation was observed between the expression of PLOD2 and CD133, PLOD2 and CD44, and PLOD2 and N-cadherin (P < .01). Moreover, a negative correlation was noted between the expression of PLOD2 and E-cadherin (P < .01). The combined expression of PLOD2 and ZEB1 were independent prognostic factors for the total survival time of patients with ESCC. CONCLUSION: PLOD2 is highly expressed in ESCC and is closely related to tumor invasion and metastasis. The mechanism of PLOD2 for promoting invasion and metastasis of ESCC may be related to activation of the EMT signaling pathway to promote EMT and tumor stem cell transformation.

PLOD2 Is a Prognostic Marker in Glioblastoma That Modulates the Immune Microenvironment and Tumor Progression.

This study aimed to investigate the role of Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase 2 (PLOD2) in glioblastoma (GBM) pathophysiology. To this end, PLOD2 protein expression was assessed by immunohistochemistry in two independent cohorts of patients with primary GBM (n1 = 204 and n2 = 203, respectively). Association with the outcome was tested by Kaplan−Meier, log-rank and multivariate Cox regression analysis in patients with confirmed IDH wild-type status. The biological effects and downstream mechanisms of PLOD2 were assessed in stable PLOD2 knock-down GBM cell lines. High levels of PLOD2 significantly associated with (p1 = 0.020; p2< 0.001; log-rank) and predicted (cohort 1: HR = 1.401, CI [95%] = 1.009−1.946, p1 = 0.044; cohort 2: HR = 1.493; CI [95%] = 1.042−2.140, p2 = 0.029; Cox regression) the poor overall survival of GBM patients. PLOD2 knock-down inhibited tumor proliferation, invasion and anchorage-independent growth. MT1-MMP, CD44, CD99, Catenin D1 and MMP2 were downstream of PLOD2 in GBM cells. GBM cells produced soluble factors via PLOD2, which subsequently induced neutrophils to acquire a pro-tumor phenotype characterized by prolonged survival and the release of MMP9. Importantly, GBM patients with synchronous high levels of PLOD2 and neutrophil infiltration had significantly worse overall survival (p < 0.001; log-rank) compared to the other groups of GBM patients. These findings indicate that PLOD2 promotes GBM progression and might be a useful therapeutic target in this type of cancer.

P3H4 and PLOD1 expression associates with poor prognosis in bladder cancer.

PURPOSE: The prolyl 3-hydroxylase family member 4 gene (P3H4) is involved in the development of human cancers. The association of P3H4 with bladder cancer (BC) prognosis is unclear. This study aimed to analyze the association of P3H4 with BC prognosis. METHODS: RNA-Seq data were downloaded from The Cancer Genome Atlas project and BC microarray datasets (GSE13507, GSE31684, and GSE32548) were downloaded from the Gene Expression Omnibus database. We analyzed the differences in P3H4 expression levels between BC tumors and non-tumor tissues and between samples with different clinical information. The association of P3H4 and P3H4-related genes with BC prognosis and the possibility of using P3H4 expression as a prognostic biomarker in BC patients were also analyzed. RevMan was used to perform the meta-analysis. RESULTS: P3H4 was upregulated in BC tissues compared with the adjacent non-tumor tissues (p = 4.06e-08). Univariate Cox regression analysis and meta-analysis showed that high P3H4 expression level contributed to a poor BC prognosis (Hazard ratio, HR = 1.348, 95% CI 1.140-1.594, p = 4.89e-04; meta-analysis: HR = 1.45, 95% CI 1.10-1.91; p = 9.00e-03). Among the genes related to P3H4, the PLOD1 gene was closely associated with P3H4 expression (r = 0.620, p = 2.49e-44). Also, a meta-analysis showed that PLOD1 expression was associated with a poor prognosis in BC patients (HR = 1.77, 95% CI 1.31-2.38; p = 2.00e-04). CONCLUSIONS: The P3H4 and PLOD1 genes might be used as reliable prognostic biomarkers for BC.

PLOD1 acts as a tumor promoter in glioma via activation of the HSF1 signaling pathway.

Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (PLOD1) is a collagen-related lysyl hydroxylase and its prognostic value in glioma patients was verified. However, its biological function in glioma has yet to be fully investigated. The PLOD1 mRNA status and clinical significance in gliomas were assessed via the GEPIA database. Overexpression or targeted depletion of PLOD1 was carried out in the human glioma cell line U87 and verified by western blotting. CCK8 and colony formation assays were implemented to examine the impact of PLOD1 on the proliferative and colony-forming phenotypes of U87 cells. Luciferase reporter assays and HSF1-specific pharmacologic inhibitors (KRIBB11) were employed to determine the regulatory relationship between PLOD1 and heat shock factor 1 (HSF1). High expression of PLOD1 was observed in tissue samples of glioblastoma multiforme (GBM) and brain lower-grade glioma (LGG). GEPIA overall survival further demonstrated that both GBM and LGG patients with high PLOD1 displayed worse clinical outcomes compared with those with low PLOD1. Overexpression and targeted depletion of PLOD1 enhanced and suppressed U87 cell proliferation and colony formation, respectively. Luciferase reporter assays showed that PLOD1 significantly enhanced the transcriptional activity of HSF1 in HEK293T cells. PLOD1 deficiency in U87 cells inhibited HSF1-induced survivin accumulation, whereas KRIBB11 also blocked the PLOD1-overexpressing induced survivin expression. An inhibitor of HSF1 signaling events abolished the increased clonogenic potential caused by PLOD1 overexpression in U87 cells. High expression of PLOD1 can increase the proliferation and colony formation of U87 cells by activating the HSF1 signaling pathway. This study suggested PLOD1/HSF1 as an effective therapeutic target for gliomas.

Lysyl hydroxylase 2 deficiency promotes filopodia formation and fibroblast migration.

Lysyl hydroxylase 2 (LH2) regulates intermolecular cross-linking of collagen molecules. Accumulation of LH2-modified collagen, which is highly stable and resistant to collagenase cleavage, is one cause of fibrosis. We previously demonstrated that conventional LH2 knockout mice showed embryonic lethality. Here we established LH2 conditional knockout mice using a tamoxifen-inducible Cre system. Morphological analysis of LH2-deficient fibroblasts by microscopy showed a dramatic increase in the number of filopodia, the finger-like cell surface projections that enable cell movement. The tips and leading edges of these filopodia exhibited up-regulated expression of Myosin-X (Myo10), a regulator of filopodial integrity. Wound healing assays demonstrated that migration of LH2-deficient cells was significantly faster than that of control cells. Gene expression profiling data also supported this phenotype. Together these findings indicate that LH2 deficiency may prevent fibrosis through decreased accumulation of LH2-cross-linked collagen, and that fibroblasts with faster migration contribute to enhanced wound healing activity. In conclusion, our cellular models provide evidence that LH2 deficiency plays a critical role in cell migration mediated through filopodia formation. Understanding the precise role of this phenotype in LH2-deficient cells may be helpful to define the pathogenesis of fibrosis. As such, detailed analyses of fibrosis and wound healing using LH2-deficient mouse models are needed.

First reported case of fragile foal syndrome type 1 in the Thoroughbred caused by PLOD1 c.2032G>A.

BACKGROUND: Warmblood Fragile Foal Syndrome Type 1 (WFFS) is an autosomal recessive disorder reported previously only in warmbloods and thought to be caused by a variant in the gene procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1, c.2032G>A, p.Gly678Arg). Given the presentation of this Thoroughbred case, we hypothesised that a similar genetic mechanism caused this phenotype. OBJECTIVES: To describe the pathological and genetic findings on a foal presenting to a veterinary practice in the UK with skin lesions similar to other Ehlers-Danlos Syndromes, including those documented for warmbloods with WFFS. STUDY DESIGN: A single case report describing a genetic investigation. METHODS: A Thoroughbred foal presenting as dystocia was euthanised for multiple skin lesions and developmental abnormalities. DNA extracted from the foal was tested for the PLOD1 variant (c.2032G>A, p.Gly678Arg) using the commercially available assay. To confirm causality and further interrogate potential novel causes of Ehlers-Danlos Syndrome, 1799 functional candidate genes, including PLOD1, were analysed using whole genome sequencing data generated from DNA extracted from the foal's muscle. These data were compared to 34 control samples from at least 11 other breeds. Variants were prioritised for further evaluation based on predicted impact on protein function. RESULTS: Post-mortem evaluation concluded that this foal suffered from a condition of collagen dysplasia. The foal was homozygous for the c.2032G>A PLOD1 variant. Only two other missense variants identified from whole genome sequencing data were also computationally predicted to be deleterious to protein function, (NPHP3 c.1253T>C, p.Leu418Pro, EPDR1 c.154G>C, p.Glu52Gln). Neither of these genes have been linked to similar phenotypes, or Ehlers-Danlos Syndrome in humans or other species and thus further investigation of these variants as the cause of EDS was not warranted. MAIN LIMITATIONS: This study is a single case report in the Thoroughbred with no additional cases from this breed yet identified to replicate this finding. CONCLUSIONS: Given the clinical presentation similar to WFFS, homozygosity for the PLOD1 variant, and absence of another more plausible causal variant from the WGS experiment, we conclude that PLOD1 c.2032G>A is the likely cause of this foal's condition. This is the first documented evidence of fragile foal syndrome caused by the PLOD1 variant in a breed outside of warmbloods, the Thoroughbred. We therefore recommend a change in the name of this disorder to fragile foal syndrome type 1 (FFS) and utilisation of genetic testing in Thoroughbreds to avoid producing affected foals.

Identification and Validation of PLOD2 as an Adverse Prognostic Biomarker for Oral Squamous Cell Carcinoma.

BACKGROUND: Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2), a key enzyme that catalyzes the hydroxylation of lysine, plays a crucial role in the progression of several solid tumors. However, its spatial expression profile and prognostic significance in oral squamous cell carcinoma (OSCC) have not been revealed. MATERIALS: Mass spectrometry was used to explore amino acid perturbations between OSCC tumor tissues and paired normal tissues of 28 patients. Then, PLOD2 mRNA and protein levels were assessed using several public databases and 18 pairs of OSCC patients' tissues. Additionally, PLOD2 spatial expression profiles were investigated in 100 OSCC patients by immunohistochemistry and its diagnostic and prognostic values were also evaluated. Lastly, gene set enrichment analysis (GSEA) was used to investigate the potential functions of PLOD2 in OSCC. RESULTS: Lysine was significantly elevated in OSCC tissues and could effectively distinguish tumor from normal tissues (AUC = 0.859, p = 0.0035). PLOD2 mRNA and protein levels were highly increased in tumor tissues of head and neck squamous cell carcinoma (HNSCC) (p < 0.001) and OSCC compared with those in nontumor tissues (p < 0.001). Histopathologically, PLOD2 was ubiquitously expressed in tumor cells (TCs) and fibroblast-like cells (FLCs) of OSCC patients but absent in tumor-infiltrating lymphocytes (TILs). Patients with highly expressed PLOD2 in TCs (PLOD2TCs) and FLCs (PLOD2FLCs) showed poor differentiation, a worse pattern of invasion (WPOI) and more lymph node metastasis (LNM), contributing to higher postoperative metastasis risk and poor survival time. However, PLOD2FLCs rather than PLOD2TCs was an independent risk factor for survival outcomes in OSCC patients. Molecularly, GSEA demonstrated highly expressed PLOD2 was mainly enriched in epithelial-mesenchymal transformation (EMT), TGF-beta signaling and hypoxia pathway, which are associated with poor clinical outcomes of OSCC patients. CONCLUSIONS: PLOD2 was a poor prognostic biomarker for OSCC patients and may affect the metastasis of OSCC through EMT pathway. These findings might shed novel sights for future research in PLOD2 targeted OSCC therapy.

Integrated Profiling Identifies PLOD3 as a Potential Prognostic and Immunotherapy Relevant Biomarker in Colorectal Cancer.

Procollagen-Lysine,2-Oxoglutarate 5-Dioxygenase 3 (PLOD3) is related to a variety of human diseases. However, its function in Colorectal cancer (CRC) remains uncertain. PLOD3 expression was analyzed using The Cancer Genome Atlas (TCGA) pan-cancer data. DAVID was used for enrichment analysis of PLOD3-related genes. The correlation between PLOD3 expression and immune cell infiltration was evaluated. Four expression profile datasets (GSE17536, GSE39582, GSE74602, and GSE113513) from Gene Expression Omnibus, and two proteomic datasets were used as validation cohorts for assessing the diagnostic and prognostic value of PLOD3 in CRC. What's more, we performed immunohistochemistry (IHC) staining for PLOD3 in 160 paired CRC specimens and corresponding adjacent non-tumor tissues. PLOD3 was highly expressed in many tumors including CRC. PLOD3 was upregulated in advanced stage CRCs, and high PLOD3 expression was associated with poor survival. High PLOD3 expression was associated with low levels of B cells, CD4+ T cells, M1 macrophages, CD8+ T cells, and multiple immunerelated characteristics. In addition, the high PLOD3 expression group had a higher TIDE score and a lower tumor mutation burden and microsatellite instability, indicating that patients with high PLOD3 expression may be resistant to immunotherapy. Additional datasets and IHC analysis were used to validate the diagnostic and prognostic value of PLOD3 at the mRNA and protein levels in CRC. Patients with non-response to immunotherapy showed increased PLOD3 expression in an immunotherapy treated dataset. PLOD3 is a potential biomarker for CRC diagnosis and prognosis prediction. CRCs with high PLOD3 expression may be resistant to immune checkpoint therapy.

Lysyl hydroxylase 1 (LH1) deficiency promotes angiotensin II (Ang II)-induced dissecting abdominal aortic aneurysm.

Rationale: The progressive disruption of extracellular matrix (ECM) proteins, particularly early elastin fragmentation followed by abnormalities in collagen fibril organization, are key pathological processes that contribute to dissecting abdominal aortic aneurysm (AAA) pathogenesis. Lysyl hydroxylase 1 (LH1) is essential for type I/III collagen intermolecular crosslinking and stabilization. However, its function in dissecting AAA has not been explored. Here, we investigated whether LH1 is significantly implicated in dissecting AAA progression and therapeutic intervention. Methods and Results: Sixteen-week-old male LH1-deficient and wild-type (WT) mice on the C57Bl/6NCrl background were infused with angiotensin II (Ang II, 1000 ng/kg per minute) via subcutaneously implanted osmotic pumps for 4 weeks. Ang II increased LH1 levels in the abdominal aortas of WT mice, whereas mice lacking LH1 developed dissecting AAA. To evaluate the related mechanism, we performed whole-transcriptomic analysis, which demonstrated that LH1 deficiency aggravated gene transcription alterations; in particular, the expression of thrombospondin-1 was markedly upregulated in the aortas of LH1-deficient mice. Furthermore, targeting thrombospondin-1 with TAX2 strongly inhibited the proinflammatory process, matrix metalloproteinase (MMP) activity and vascular smooth muscle cells (VSMCs) apoptosis, ultimately decreasing the incidence of dissecting AAA. Restoration of LH1 protein expression in LH1-deficient mice by intraperitoneal injection of an adeno-associated virus normalized thrombospondin-1 levels, subsequently alleviating dissecting AAA formation and preserving aortic structure and function. Consistently, in human AAA specimens, decreased LH1 expression was associated with increased thrombospondin-1 levels. Conclusions: LH1 deficiency contributes to dissecting AAA pathogenesis, at least in part, by upregulating thrombospondin-1 expression, which subsequently enables proinflammatory processes, MMP activation and VSMCs apoptosis. Our study provides evidence that LH1 is a potential critical therapeutic target for AAA.

FK506 binding protein 10: a key actor of collagen crosslinking in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common type of malignant tumor in the kidney. With numbers of patients whose physical condition or tumor stage not suitable for radical surgery, they only have a narrow choice of using VEGF/mTOR targeted drugs to control their tumors, but ccRCC often shows resistance to these drugs. Therefore, identifying a new therapeutic target is of urgent necessity. In this study, for the first time, we concluded from bioinformatics analyses and in vitro research that FK506 binding protein 10 (FKBP10), together with its molecular partner Lysyl hydroxylase 2 (LH2/PLOD2), participate in the process of type I collagen synthesis in ccRCC via regulating crosslinking of pro-collagen chains. Our findings may provide a potential therapeutic target to treat ccRCC in the future.