RESUMO
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%).
Assuntos
Rabdomiossarcoma , Fatores de Transcrição , Adulto Jovem , Criança , Humanos , Adulto , Adolescente , Pessoa de Meia-Idade , Idoso , Idoso de 80 Anos ou mais , Hibridização in Situ Fluorescente , Estudos Retrospectivos , Fatores de Transcrição/genética , Proteína EWS de Ligação a RNA/genética , Rabdomiossarcoma/genética , Rabdomiossarcoma/patologia , Fusão Gênica , Biomarcadores Tumorais/genética , Proteínas de Ligação a RNA/genética , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética , Proteínas de Ligação a DNA/genéticaRESUMO
The tumor microenvironment (TME) plays a vital role in tumor invasion and metastasis and provides a rich environment for identifying novel therapeutic targets. The TME landscape consists of an extracellular matrix (ECM) and stromal cells. ECM is a major component of TME that mediates the interaction between cancer cells and stromal cells to promote invasion and metastasis. We have shown in published work that RASSF1C promotes cancer stem cell development, migration, and drug resistance, in part, by promoting EMT through a mechanism that involves up-regulation of the PIWIL1-piRNA axis. Consistent with this, in this study, we demonstrate that RASSF1C promotes lung cancer metastasis in vivo using an orthotopic mouse model. Interestingly, two target genes identified in a previously conducted microarray study to be up-regulated by RASSF1C in breast and non-small cell lung cancer (NSCLC) cells are prolyl 4-hydroxylase alpha-2 (P4HA2) and procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2). In cancer, P4H2A and PLOD2 are vital for collagen posttranslational modification and folding leading to the formation of a stiff ECM and induction of EMT and cancer stem cell marker gene expression, resulting in metastatic dissemination. Here, we also show that overexpression of RASSF1C up-regulates Collagen I, P4HA2, and PLOD2 in vitro. Up-regulation of P4HA2 and PLOD2 by RASSF1C was also confirmed in lung and breast cancer cells in vivo using mouse models. Further, we found that treatment of wildtype lung cancer cells or lung cancer cells overexpressing RASSF1C or PIWIL1 with piR-35127 and 46545 (both down-regulated by RASSF1C) decreased lung cancer cell invasion/migration. Taken together, our findings suggest that RASSF1C may promote lung cancer cell ECM remodeling to induce lung cancer cell stemness, invasion, and metastasis, in part, by up-regulating a previously unknown PIWIL1-P4HA2-PLOD2 pathway. Furthermore, piR-35127 and piR-46545 could potentially be important anti-metastatic tools.
RESUMO
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.
Assuntos
Neoplasias Ósseas , Neoplasias Colorretais , Neoplasias do Endométrio , Feminino , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Neoplasias Colorretais/genética , Linhagem Celular Tumoral , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Proteases Específicas de Ubiquitina/metabolismoRESUMO
BACKGROUND: In recent years, N6-methyladenosine (m6A) methylation modification of mRNA has been studied extensively. It has been reported that m6A determines mRNA fate and participates in many cellular functions and reactions, including oxidative stress. The PLOD2 gene encodes a protein that plays a key role in tissue remodeling and fibrotic processes. METHODS: The m6A methylation and expression levels of PLOD2 were determined by m6A methylated RNA immunoprecipitation sequencing (MeRIP-seq) and MeRIP-quantitative polymerase chain reaction (qPCR) in the testes of varicocele rats compared with control. To determine whether IGF2BP2 had a targeted effect on the PLOD2 mRNA, RNA immunoprecipitation-qPCR (RIP-qPCR) and luciferase assays were performed. CRISPR/dCas13b-ALKBH5 could downregulate m6A methylation level of PLOD2, which plays an important role in PLOD2-mediated cell proliferation and apoptosis in GC-2 cells. RESULTS: PLOD2 was frequently exhibited with high m6A methylation and expression level in the testes of varicocele rats compared with control. In addition, we found that IGF2BP2 binds to the m6A-modified 3' untranslated region (3'-UTR) of PLOD2 mRNA, thereby positively regulating its mRNA stability. Targeted specific demethylation of PLOD2 m6A by CRISPR/dCas13b-ALKBH5 system can significantly decrease the m6A and expression level of PLOD2. Furthermore, demethylation of PLOD2 mRNA dramatically promote GC-2 cell proliferation and inhibit cell apoptosis under oxidative stress. CONCLUSION: As a result, we found that varicocele-induced oxidative stress promoted PLOD2 expression level via m6A methylation modification. In addition, targeting m6A demethylation of PLOD2 by CRISPR/dCas13b-ALKBH5 system can regulate GC-2 cell proliferation and apoptosis under oxidative stress. Taken together, our study has acquired a better understanding of the mechanisms underlying male infertility associated with oxidative stress, as well as a novel therapeutic target for male infertility.
Assuntos
Infertilidade Masculina , Varicocele , Masculino , Animais , Ratos , Humanos , Espermatócitos , Regiões 3' não Traduzidas , Adenosina , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase , Proteínas de Ligação a RNARESUMO
This study explores the effect of apigenin(APG), oxymatrine(OMT), and APG+OMT on the proliferation of non-small cell lung cancer cell lines and the underlying mechanisms. Cell counting kit-8(CCK-8) assay was used to detect the vitality of A549 and NCI-H1975 cells, and colony formation assay to evaluate the colony formation ability of the cells. EdU assay was employed to examine the proliferation of NCI-H1975 cells. RT-qPCR and Western blot were performed to detect the mRNA and protein expression of PLOD2. Molecular docking was carried out to explore the direct action ability and action sites between APG/OMT and PLOD2/EGFR. Western blot was used to study the expression of related proteins in EGFR pathway. The viability of A549 and NCI-H1975 cells was inhibited by APG and APG+OMT at 20, 40, and 80 µmol·L~(-1) in a dose-dependent manner. The colony formation ability of NCI-H1975 cells was significantly suppressed by APG and APG+OMT. The mRNA and protein expression of PLOD2 was significantly inhibited by APG and APG+OMT. In addition, APG and OMT had strong binding activity with PLOD2 and EGFR. In APG and APG+OMT groups, the expression of EGFR and proteins in its downstream signaling pathways was significantly down-regulated. It is concluded that APG in combination with OMT could inhibit non-small lung cancer, and the mechanism may be related to EGFR and its downstream signaling pathways. This study lays a new theoretical basis for the clinical treatment of non-small cell lung cancer with APG in combination with OMT and provides a reference for further research on the anti-tumor mechanism of APG in combination with OMT.
Assuntos
Alcaloides , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Apigenina , Simulação de Acoplamento Molecular , Quinolizinas , RNA Mensageiro , Receptores ErbBRESUMO
Bruck Syndrome (BS) is a very rare disorder characterized by osteogenesis imperfecta (OI) associated with congenital contractures and is caused by mutations in FKBP10 or PLOD2 genes. Herein, we describe 13 patients from 9 unrelated Egyptian families with BS. All patients had white sclerae, recurrent fractures, kyphoscoliosis and osteoporosis with variable degrees of severity. Large joint contractures were seen in 11 patients, one patient had contractures of small interphalangeal joints, and one patient had no contractures. Unusual findings noted in individual patients included microcephaly, dental malocclusion, enamel hypoplasia, unilateral congenital dislocation of knee joint, prominent tailbone, and myopathy. Nine different variants were identified in FKBP10 and PLOD2 including five novel ones. FKBP10 variants were found in six families (67%) while PLOD2 variants were identified in three families (33%). The four families, with two affected sibs each, showed inter- and intrafamilial phenotypic variability. In conclusion, we report five novel variants in FKBP10 and PLOD2 thus, expanding the mutational spectrum of BS. In addition, our results expand the phenotypic spectrum, describe newly associated orodental findings, and further illustrate the phenotypic overlap between OI and Bruck syndrome supporting the suggestion of considering BS as a variant of OI rather than a separate entity.
Assuntos
Artrogripose , Contratura , Anormalidades Musculoesqueléticas , Osteogênese Imperfeita , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase , Proteínas de Ligação a Tacrolimo , Artrogripose/diagnóstico , Artrogripose/genética , Contratura/genética , Humanos , Anormalidades Musculoesqueléticas/genética , Mutação , Osteogênese Imperfeita/complicações , Osteogênese Imperfeita/genética , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética , Proteínas de Ligação a Tacrolimo/genéticaRESUMO
INTRODUCTION: Bruck syndrome is a rare autosomal recessive disease characterized by multiple joint contractures, bone fragility, and fractures. Two genes have been associated with Bruck syndrome, FKBP10 and PLOD2, though they are phenotypically indistinguishable. CASE PRESENTATION: We present a prenatally diagnosed case of Bruck syndrome in a young multiparous woman, with no notable personal, family or obstetric history. A 12-week ultrasound raised the suspicion of short long bones, subsequently confirmed at 16 weeks. In addition, bilateral fixed flexion of the elbow, wrist, and knee joints as well as talipes was observed. Chromosomal SNP microarray analysis (0.2 Mb) detected a homozygous deletion at chromosome 3, band q24, involving a part of PLOD2 to a part of PLSCR4. At mid-trimester morphology, bilateral intrauterine fractures of the humerus and femur were evident. In the late third trimester, a fetal echocardiogram noted enlargement of the right heart with severe tricuspid regurgitation in combination with pulmonary insufficiency and a restrictive arterial duct. The potential risk of premature closure of the ductus arteriosus near term led to delivery by emergency caesarean section. CONCLUSION: To our knowledge, this is the first case of Bruck syndrome prenatally confirmed by chromosomal microarray analysis and the second reported case with an extra-skeletal abnormality. This case highlights the importance of comprehensive fetal morphological assessment during pregnancy as diagnosis of an additional abnormality has the potential to impact both management and prognosis.
Assuntos
Artrogripose , Osteogênese Imperfeita , Humanos , Gravidez , Feminino , Artrogripose/complicações , Artrogripose/diagnóstico , Artrogripose/genética , Homozigoto , Cesárea , Deleção de Sequência , Osteogênese Imperfeita/complicações , Osteogênese Imperfeita/diagnóstico , Osteogênese Imperfeita/genética , Proteínas de Transferência de Fosfolipídeos/genéticaRESUMO
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.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Humanos , Imuno-Histoquímica , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Prognóstico , Microambiente TumoralRESUMO
The overactivation of Wnt/ß-catenin signaling is a hallmark of colorectal cancer (CRC) development. We identified the cell adhesion molecule L1CAM (L1) as a target of ß-catenin-TCF transactivation in CRC cells. The overexpression of L1 in CRC cells confers enhanced proliferation, motility, tumorigenesis and liver metastasis, and L1 is exclusively localized in the invasive areas of human CRC tissue. A number of genes are induced after L1 transfection into CRC cells by a mechanism involving the cytoskeletal protein ezrin and the NF-κB pathway. When studying the changes in gene expression in CRC cells overexpressing L1 in which ezrin levels were suppressed by shRNA to ezrin, we discovered the collagen-modifying enzyme lysyl hydroxylase 2 (PLOD2) among these genes. We found that increased PLOD2 expression was required for the cellular processes conferred by L1, including enhanced proliferation, motility, tumorigenesis and liver metastasis, since the suppression of endogenous PLOD2 expression, or its enzymatic activity, blocked the enhanced tumorigenic properties conferred by L1. The mechanism involved in increased PLOD2 expression by L1 involves ezrin signaling and PLOD2 that affect the SMAD2/3 pathway. We found that PLOD2 is localized in the colonic crypts in the stem cell compartment of the normal mucosa and is found at increased levels in invasive areas of the tumor and, in some cases, throughout the tumor tissue. The therapeutic strategies to target PLOD2 expression might provide a useful approach for CRC treatment.
Assuntos
Neoplasias do Colo/patologia , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Colágeno/metabolismo , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Proteínas do Citoesqueleto/metabolismo , Regulação Neoplásica da Expressão Gênica , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/secundário , Masculino , Camundongos Nus , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Collagens are extracellular matrix (ECM) proteins that support the structural and biomechanical integrity of many tissues. Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) encodes the only lysyl hydroxylase (LH) isoform that specifically hydroxylates lysine residues in collagen telopeptides, a post-translational modification required for the formation of stabilized cross-links. PLOD2 expression is induced by hypoxia and transforming growth factor-ß1 (TGF-ß1), well-known stimuli for the formation of a fibrotic ECM, which can lead to pathological fibrosis underlying several diseases. Here, using human and murine fibroblasts, we studied the molecular determinants underlying hypoxia- and TGF-ß1-induced PLOD2 expression and its impact on collagen biosynthesis. Deletion mapping and mutagenesis analysis identified specific binding sites for hypoxia-inducible factors (HIF) and TGF-ß1-activated SMAD proteins on the human PLOD2 gene promoter that were required for these stimuli to induce PLOD2 expression. Interestingly, our experiments also revealed that HIF signaling plays a preponderant role in the SMAD pathway, as intact HIF sites were absolutely required for TGF-ß1 to exert its effect on SMAD-binding sites. We also found that silencing PLOD2 expression did not alter soluble collagen accumulation in the extracellular medium, but it effectively abolished the deposition into the insoluble collagen matrix. Taken together, our findings reveal the existence of a hierarchical relationship between the HIF and SMAD signaling pathways for hypoxia- and TGF-ß1-mediated regulation of PLOD2 expression, a key event in the deposition of collagen into the ECM.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fator 1 Induzível por Hipóxia/metabolismo , Oxigênio/metabolismo , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética , Proteínas Smad/metabolismo , Células 3T3 , Animais , Hipóxia Celular , Linhagem Celular Tumoral , Matriz Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Camundongos , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Regiões Promotoras Genéticas , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
The purpose of this study was to characterize the expression of procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2), a membrane-bound homodimeric enzyme that specifically hydroxylates lysine in the telopeptide of procollagens, and assess the clinical significance of PLOD2 in colorectal cancer (CRC). Our results show that PLOD2 is highly expressed in CRC tumor tissues and cell lines, both at the mRNA and protein levels. Next, we found that PLOD2 was positively correlated with tumor grade (P = 0.001), T stage (P = 0.001), N stage (P < 0.001), and an advanced TNM stage (P < 0.001). Knockdown of PLOD2 attenuated CRC cell proliferation, migration, and invasiveness, in vitro. Our analysis of the mechanism behind the effects of PLOD2 suggests that PLOD2 affected glycolysis by regulating the expression of hexokinase 2 (HK2). HK2 reverses the inhibitory effects of PLOD2 knockdown in CRC. Furthermore, the data suggest that PLOD2 regulates the expression of HK2 via the STAT3 signaling pathway. Survival analysis revealed that high expression levels of PLOD2 (HR = 3.800, P < 0.001) and HK2 expression (HR = 10.222, P < 0.001) correlated with the overall survival rate. After analyzing their expression and correlation, PLOD2 positively correlated with HK2 (r = 0.590, P < 0.001). Our findings have revealed that PLOD2 is a novel regulatory factor in glucose metabolism, exerted via controlling HK2 expression in CRC cells, suggesting PLOD2 as a promising therapeutic target for CRC treatment.
Assuntos
Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão Gênica , Glicólise , Hexoquinase/metabolismo , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Regulação para Cima , Aerobiose , Idoso , Células CACO-2 , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Colágeno/química , Progressão da Doença , Feminino , Glucose/metabolismo , Células HT29 , Humanos , Lisina/química , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , RNA Mensageiro/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de SinaisRESUMO
Epigenetic editing, an emerging technique used for the modulation of gene expression in mammalian cells, is a promising strategy to correct disease-related gene expression. Although epigenetic reprogramming results in sustained transcriptional modulation in several in vivo models, further studies are needed to develop this approach into a straightforward technology for effective and specific interventions. Important goals of current research efforts are understanding the context-dependency of successful epigenetic editing and finding the most effective epigenetic effector(s) for specific tasks. Here we tested whether the fibrosis- and cancer-associated PLOD2 gene can be repressed by the DNA methyltransferase M.SssI, or by the non-catalytic Krüppel associated box (KRAB) repressor directed to the PLOD2 promoter via zinc finger- or CRISPR-dCas9-mediated targeting. M.SssI fusions induced de novo DNA methylation, changed histone modifications in a context-dependent manner, and led to 50%-70% reduction in PLOD2 expression in fibrotic fibroblasts and in MDA-MB-231 cancer cells. Targeting KRAB to PLOD2 resulted in the deposition of repressive histone modifications without DNA methylation and in almost complete PLOD2 silencing. Interestingly, both long-term TGFß1-induced, as well as unstimulated PLOD2 expression, was completely repressed by KRAB, while M.SssI only prevented the TGFß1-induced PLOD2 expression. Targeting transiently expressed dCas9-KRAB resulted in sustained PLOD2 repression in HEK293T and MCF-7 cells. Together, these findings point to KRAB outperforming DNA methylation as a small potent targeting epigenetic effector for silencing TGFß1-induced and uninduced PLOD2 expression.
Assuntos
Inativação Gênica , Heterocromatina/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética , Adulto , Células Cultivadas , DNA-Citosina Metilases/genética , DNA-Citosina Metilases/metabolismo , Epigênese Genética , Células HEK293 , Humanos , Fatores de Transcrição Kruppel-Like/genética , Células MCF-7 , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Regiões Promotoras Genéticas , Ativação Transcricional , Fator de Crescimento Transformador beta/metabolismoRESUMO
BACKGROUND: Advanced stage laryngeal squamous cell carcinoma (LSCC) presents a poor prognosis; thus, there is a great need to identify novel prognostic molecular markers. Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) is thought to be a novel prognostic factor in several cancers, but its role in LSCC remains unknown. Cancer stem cells (CSCs) are responsible for most instances of tumor recurrence and the development of drug resistance and have been proven to be present in head and neck cancers. Our preliminary study indicated that PLOD2 was elevated in LSCC tissues; therefore, we hypothesized that PLOD2 is related to the prognosis of LSCC patients and aimed to explore the role and underlying mechanism of PLOD2 in LSCC. METHODS: We validated the prognostic role of PLOD2 in 114 LSCC patients by immunohistochemistry. Stable PLOD2-overexpressing Hep-2 and FaDu cells were established and assessed by molecular biology and biochemistry methods both in vitro and in vivo. RESULTS: We confirmed that PLOD2 overexpression was correlated with poor prognosis in LSCC patients. PLOD2 overexpression strengthened the CSC-like properties of Hep-2 and FaDu cells, activated the Wnt signaling pathway and conferred drug resistance in LSCC in vitro and in vivo. CONCLUSIONS: We found that PLOD2 could serve as a prognostic marker in patients with LSCC and confer drug resistance in LSCC by increasing CSC-like traits; in addition, a Wnt-responsive CSC pathway was identified.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Laríngeas/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antineoplásicos/farmacologia , Biomarcadores Tumorais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Laríngeas/metabolismo , Neoplasias Laríngeas/mortalidade , Neoplasias Laríngeas/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Prognóstico , Via de Sinalização Wnt , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Breast cancer cells recruit surrounding stromal cells, such as cancer-associated fibroblasts (CAFs), to remodel collagen and promote tumor metastasis. Adipocytes are the most abundant stromal partners in breast tissue, local invasion of breast cancer leads to the proximity of cancer cells and adipocytes, which respond to generate cancer-associated adipocytes (CAAs). These cells exhibit enhanced secretion of extracellular matrix related proteins, including collagens. However, the role of adipocyte-derived collagen on breast cancer progression still remains unclear. METHODS: Adipocytes were cocultured with breast cancer cells for 3D collagen invasion and collagen organization exploration. Breast cancer cells and adipose tissue co- implanted mouse model, clinical breast cancer samples analysis were used to study the crosstalk between adipose and breast cancer cells in vivo. A combination of proteomics, enzyme-linked immunosorbent assay, loss of function assay, qPCR, western blot, database analysis and chromatin immunoprecipitation assays were performed to study the mechanism mediated the activation of PLOD2 in adipocytes. RESULTS: It was found that CAAs remodeled collagen alignment during crosstalk with breast cancer cells in vitro and in vivo, which further promoted breast cancer metastasis. Tumor-derived PAI-1 was required to activate the expression of the intracellular enzyme procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) in CAAs. Pharmacologic blockade of PAI-1 or PLOD2 disrupted the collagen reorganization in CAAs. Mechanistically, it was observed that PI3K/AKT pathway was activated in adipocytes upon co-culturing with breast cancer cells or treatment with recombinant PAI-1, which could promote the translocation of transcription factor FOXP1 into the nucleus and further enhanced the promoter activity of PLOD2 in CAAs. In addition, collagen reorganization at the tumor-adipose periphery, as well as the positive relevance between PAI-1 and PLOD2 in invasive breast carcinoma were confirmed in clinical specimens of breast cancer. CONCLUSION: In summary, our findings revealed a new stromal collagen network that favors tumor invasion and metastasis establish between breast cancer cells and surrounding adipocytes at the tumor invasive front, and identified PLOD2 as a therapeutic target for metastatic breast cancer treatment.
Assuntos
Adipócitos/metabolismo , Neoplasias da Mama/metabolismo , Colágeno/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Células 3T3 , Adulto , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Fatores de Transcrição Forkhead/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Metástase Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Objective: To investigate the relationship of procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2) expression and the clinical characteristics of osteosarcoma, and explore the potential mechanism of tumour metastasis promoted by PLOD2. Methods: The expression of PLOD2 in osteosarcoma tissues and paired adjacent tissues were detected by immunohistochemistry and qRT-PCR. Correlation of PLOD2 expression in osteosarcoma with the clinical pathologic features was analyzed by Chi square test and Kaplan-Meier analysis.Fibrillar collagen formation and collagen deposition in the tumor tissues were detected by picrosirius red staining. We transfected U-2OS cells with LV-vector, LV-over/PLOD2, sh-NC and sh-PLOD2. The expression of PLOD2 was detected by qRT-PCR. The impact of POLD2 on U-2OS cell invasion was determined by wound-healing assay and Transwell migration assay. The expressions of PLOD2/FAK/JAK2-STAT3 signal pathway related proteins were detected by western blotting. Results: The high expression level of PLOD2 in osteosarcoma tissues was 72.5%, significantly higher than 0% in paired adjacent noncancerous tissues (P<0.01), the expression of PLOD2 was positively correlated with lymph node metastasis, pulmonary metastasis and poor outcome (P<0.01). The same results were also observed in qRT-PCR assay. The median survival time of patients with high expression of PLOD2 protein was 13 months, significantly shorter than 32 months of patients with low expression of PLOD2 (P<0.05). The result of picrosirius red staining showed that the percentage of collagen fiber deposition in the osteosarcoma tissue with high level of PLOD2 was (74.43+ 9.63)%, significantly higher than (9.67±1.28)% in tissue with low expression of PLOD2 (P<0.001). The result of wound-healing and Transwell migration assay showed that over-expression of PLOD2 markedly promoted the invasion, however, knockdown of PLOD2 suppressed the invasion of U-2OS cells (both P<0.01). The result of western blotting showed that over-expression of PLOD2 significantly increased the expression levels of p-FAK, p-JAK2, p-STAT3, but knockdown PLOD2 decreased the levels of p-FAK, p-JAK2, p-STAT3 in U-2OS cells. Conclusions: Up-regulation of PLOD2 in osteosarcoma is correlated with lymphatic and distant metastasis. PLOD2 promotes invasion and metastasis of osteosarcoma might through FAK/JAK2-STAT3 signal pathway.
Assuntos
Neoplasias Ósseas/patologia , Osteossarcoma/patologia , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Regulação para Cima , Humanos , Invasividade Neoplásica , Metástase NeoplásicaRESUMO
Peritoneal dissemination is the most common metastatic pattern in advanced gastric cancer (GC) and has a very poor prognosis. However, its molecular mechanism has not been elucidated. Our study investigated genes associated with peritoneal dissemination of GC. We performed combined expression analysis of metastatic GC cell lines and identified Procollagen-lysine, 2-oxoglutarate 5-dioxygenase2 (PLOD2) as a potential regulator of peritoneal dissemination. PLOD2 is regulated by hypoxia-inducible factor-1 (HIF-1) and mediates extracellular matrix remodeling, alignment, and mechanical properties. We analyzed PLOD2 expression immunohistochemically in 179 clinical samples, and found high PLOD2 expression to be significantly associated with peritoneal dissemination, leading to poor prognosis. In an in vivo-collected metastatic cell line, downregulation of PLOD2 by siRNA reduced invasiveness and migration. Hypoxia upregulated PLOD2 mediated by HIF-1, and promoted invasiveness and migration. After exposure to hypoxia, a cell line transfected with siPLOD2 exhibited significantly suppressed invasiveness and migration, despite high HIF-1 expression. These findings indicate that PLOD2 is a regulator of, and candidate therapeutic target for peritoneal dissemination of GC. Although peritoneal dissemination of GC has a very poor prognosis, its molecular mechanism has not been elucidated. We identified PLOD2 regulated by HIF-1 as a potential regulator of peritoneal dissemination of GC. Finally, we showed that PLOD2 promotes cell invasiveness and migration in GC under hypoxia and lead to peritoneal dissemination of GC.
Assuntos
Biomarcadores Tumorais/metabolismo , Movimento Celular , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Recidiva Local de Neoplasia/patologia , Neoplasias Peritoneais/secundário , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Neoplasias Gástricas/patologia , Idoso , Biomarcadores Tumorais/genética , Hipóxia Celular , Feminino , Seguimentos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Metástase Linfática , Masculino , Invasividade Neoplásica , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/metabolismo , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/metabolismo , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética , Prognóstico , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Taxa de Sobrevida , Células Tumorais CultivadasRESUMO
Bruck syndrome is a rare autosomal recessive form of osteogenesis imperfecta (OI), which is mainly characterized by joint contractures and recurrent fragility fractures. Mutations in FKBP10 and PLOD2 were identified as the underlying genetic defects of Bruck syndrome. Here we investigated the phenotypes and the pathogenic mutations of three unrelated Chinese patients with Bruck syndrome. Clinical fractures, bone mineral density (BMD), bone turnover biomarkers, and skeletal images were evaluated in detail. The pathogenic mutations were identified by targeted next-generation sequencing and subsequently confirmed by Sanger sequencing and cosegregation analysis. We also evaluated the effects of zoledronic acid on bone fracture incidence and BMD of the patients. Three patients had congenital joint contractures, recurrent fragility fractures, camptodactyly, clubfoot, scoliosis, but without dentinogenesis imperfecta and hearing loss. Five novel heterozygous mutations were detected in PLOD2, including three heterozygous missense mutations (c.1138C>T, p.Arg380Cys; c.1153T>C, p.Cys385Arg; and c.1982G>A, p.Gly661Asp), one heterozygous nonsense mutation (c.2038C>T, p.Arg680X), and one heterozygous splice-site mutation (c.503-2A>G). Their parents were all heterozygous carriers of these mutations in PLOD2. No clear genotype-phenotype correlations were found in these patients with PLOD2 mutations. Z-score of BMD was significantly increased, but scoliosis progressed and new bone fractures occurred during the treatment of zoledronic acid. Our findings expanded the spectrum of gene mutations of Bruck syndrome.
Assuntos
Artrogripose/genética , Mutação/genética , Osteogênese Imperfeita/genética , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética , Densidade Óssea/genética , Criança , Pré-Escolar , Feminino , Fraturas Ósseas/genética , Estudos de Associação Genética/métodos , Heterozigoto , Humanos , Masculino , FenótipoRESUMO
BACKGROUND: Adipocytes make up the major component of breast tissue, accounting for 90% of stromal tissue. Thus, the crosstalk between adipocytes and breast cancer cells may play a critical role in cancer progression. Adipocyte-breast cancer interactions have been considered important for the promotion of breast cancer metastasis. However, the specific mechanisms underlying these interactions are unclear. In this study, we investigated the mechanisms of adipocyte-mediated breast cancer metastasis. METHODS: Breast cancer cells were cocultured with mature adipocytes for migration and 3D matrix invasion assays. Next, lentivirus-mediated loss-of-function experiments were used to explore the function of lysyl hydroxylase (PLOD2) in breast cancer migration and adipocyte-dependent migration of breast cancer cells. The role of PLOD2 in breast cancer metastasis was further confirmed using orthotopic mammary fat pad xenografts in vivo. Clinical samples were used to confirm that PLOD2 expression is increased in tumor tissue and is associated with poor prognosis of breast cancer patients. Cells were treated with cytokines and pharmacological inhibitors in order to verify which adipokines were responsible for activation of PLOD2 expression and which signaling pathways were activated in vitro. RESULTS: Gene expression profiling and Western blotting analyses revealed that PLOD2 was upregulated in breast cancer cells following coculture with adipocytes; this process was accompanied by enhanced breast cancer cell migration and invasion. Loss-of-function studies indicated that PLOD2 knockdown suppressed cell migration and disrupted the formation of actin stress fibers in breast cancer cells and abrogated the migration induced by following coculture with adipocytes. Moreover, experiments performed in orthotopic mammary fat pad xenografts showed that PLOD2 knockdown could reduce metastasis to the lung and liver. Further, high PLOD2 expression correlated with poor prognosis of breast cancer patients. Mechanistically, adipocyte-derived interleukin-6 (IL-6) and leptin may facilitate PLOD2 upregulation in breast cancer cells and promote breast cancer metastasis in tail vein metastasis assays. Further investigation revealed that adipocyte-derived IL-6 and leptin promoted PLOD2 expression through activation of the JAK/STAT3 and PI3K/AKT signaling pathways. CONCLUSIONS: Our study reveals that adipocyte-derived IL-6 and leptin promote PLOD2 expression by activating the JAK/STAT3 and PI3K/AKT signaling pathways, thus promoting breast cancer metastasis.
Assuntos
Adipócitos/metabolismo , Neoplasias da Mama/patologia , Interleucina-6/metabolismo , Leptina/metabolismo , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Regulação para Cima , Células 3T3-L1 , Adipocinas/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Feminino , Técnicas de Silenciamento de Genes , Humanos , Janus Quinases/metabolismo , Camundongos , Metástase Neoplásica , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/deficiência , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética , Prognóstico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Microambiente TumoralRESUMO
BACKGROUND: Intra-tumoral hypoxia and increases in extracellular level of transforming growth factor ß1 (TGF-ß1), which are common findings in cancer, are associated with an increased risk of metastasis and mortality. Moreover, metastasis is the leading cause of death of patients with cervical cancer. PLOD2 is an intracellular enzyme required for the biogenesis of collagen and its expression can be induced by hypoxia and TGF-ß1. Specifically, PLOD2 is up-regulated in several types of cancer, including cervical cancer, and is associated with cancer metastasis. Thus, in this research, we aimed to investigate the role of PLOD2 in the motility of cervical cancer cells and to show the molecular mechanism underlying this effect. METHODS: siRNA was used to knockdown PLOD2 in the cervical cancer cell lines HeLa and SiHa. The ability of cells to migrate and invade, their adhesion to type I collagen, and their capacity for epithelial-to-mesenchymal transition (ΕΜΤ) and focal adhesion formation were analyzed. Gene expression changes were validated by qRT-PCR, Western blotting and Immunocytochemistry. The morphological status of cells was examined using phalloidin staining. Differences in PLOD2 expression among patients with cervical cancer were identified by referring to public databases, including Oncomine and TCGA. RESULTS: Hypoxia and TGF-ß1 enhanced the expression of PLOD2 in HeLa and SiHa cells, and knockdown of PLOD2 inhibited cell motility and EMT. Moreover, the depletion of PLOD2 attenuated hypoxia-mediated cell migration and invasion and inhibited TGF-ß1-induced phenotypic EMT-like changes by preventing ß-catenin from entering the nucleus. In addition, PLOD2 depletion decreased cell adhesion to extracellular collagen by inhibiting the formation of focal adhesions. Moreover, a database analysis showed that PLOD2 expression is associated with human cervical cancer progression. CONCLUSIONS: Overall, our results indicated that hypoxia- and TGF-ß1-induced PLOD2 expression promotes the migratory, invasive and adhesive capacities of cervical cancer cells by participating in TGF-ß1 induced EMT and the formation of focal adhesions.
RESUMO
Objective: To investigate associations between obesity-linked systemic factors and gene expression indicative for the inflammatory and fibrotic processes in the infrapatellar fat pad (IFP), in a population of obese patients with end-stage knee osteoarthritis (KOA). Methods: We collected human IFPs from 48 patients with a mean body mass index (BMI) of 35.44 âkg/m2 during total knee replacement procedures. These patients were part of a randomized controlled trial and met the criteria of having OA and a BMI of ≥30 âkg/m2. Blood samples were collected to assess serum levels of glucose, total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, and leptin. Total body composition was measured using dual-energy X-ray absorptiometry. Gene expressions of IL6, TNFA, COL1A1, IL1B, ASMA, PLOD2 in the IFP were analyzed. Results: Univariate analysis resulted in a positive correlation between BMI and procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 (PLOD2) expression (r2 â= â0.13). In univariate analyses of obesity-linked systemic factors and PLOD2, significant correlations were found for lean mass (r2 â= â0.20), fat mass (r2 â= â0.20), serum cholesterol (r2 â= â0.17), serum triglycerides (r2 â= â0.19) and serum leptin (r2 â= â0.10). A multiple linear regression model indicated fat mass to be a strong predictor of PLOD2 production in the IFP (r2 â= â0.22, P â= â0.003). Conclusion: Our study demonstrates the positive association between fat mass and PLOD2 expression in the IFP of obese end-stage knee OA patients. This may indicate that within this patient population the fibrotic process in the IFP is influenced by systemic adipose tissue, next to local inflammatory processes.