RESUMO
Skeletal growth promoted by endochondral ossification is tightly coordinated by self-renewal and differentiation of chondrogenic progenitors. Emerging evidence has shown that multiple skeletal stem cells (SSCs) participate in cartilage formation. However, as yet, no study has reported the existence of common long-lasting chondrogenic progenitors in various types of cartilage. Here, we identify Gli1+ chondrogenic progenitors (Gli1+ CPs), which are distinct from PTHrP+ or FoxA2+ SSCs, are responsible for the lifelong generation of chondrocytes in the growth plate, vertebrae, ribs, and other cartilage. The absence of Gli1+ CPs leads to cartilage defects and dwarfishness phenotype in mice. Furthermore, we show that the BMP signal plays an important role in self-renewal and maintenance of Gli1+ CPs. Deletion of Bmpr1α triggers Gli1+ CPs quiescence exit and causes the exhaustion of Gli1+ CPs, consequently disrupting columnar cartilage. Collectively, our data demonstrate that Gli1+ CPs are common long-term chondrogenic progenitors in multiple types of cartilage and are essential to maintain cartilage homeostasis.
Assuntos
Cartilagem , Condrogênese , Animais , Camundongos , Proteína GLI1 em Dedos de Zinco/genética , Condrogênese/genética , Condrócitos , Osteogênese , Diferenciação CelularRESUMO
The development of musculoskeletal tissues such as tendon, enthesis, and bone relies on proliferation and differentiation of mesenchymal progenitor cells. Gli1+ cells have been described as putative stem cells in several tissues and are presumed to play critical roles in tissue formation and maintenance. For example, the enthesis, a fibrocartilage tissue that connects tendon to bone, is mineralized postnatally by a pool of Gli1+ progenitor cells. These cells are regulated by hedgehog signaling, but it is unclear if TGFß signaling, necessary for tenogenesis, also plays a role in their behavior. To examine the role of TGFß signaling in Gli1+ cell function, the receptor for TGFß, TbR2, was deleted in Gli1-lineage cells in mice at P5. Decreased TGFß signaling in these cells led to defects in tendon enthesis formation by P56, including defective bone morphometry underlying the enthesis and decreased mechanical properties. Immunohistochemical staining of these Gli1+ cells showed that loss of TGFß signaling reduced proliferation and increased apoptosis. In vitro experiments using Gli1+ cells isolated from mouse tail tendons demonstrated that TGFß controls cell proliferation and differentiation through canonical and non-canonical pathways and that TGFß directly controls the tendon transcription factor scleraxis by binding to its distant enhancer. These results have implications in the development of treatments for tendon and enthesis pathologies.
Assuntos
Proteínas Hedgehog , Fator de Crescimento Transformador beta , Animais , Camundongos , Proteínas Hedgehog/genética , Proteína GLI1 em Dedos de Zinco/genética , Tendões , Transdução de SinaisRESUMO
BACKGROUND: Breast cancer, the most prevalent cancer in women worldwide, faces treatment challenges due to drug resistance, posing a serious threat to patient survival. The present study aimed to identify the key molecules that drive drug resistance and aggressiveness in breast cancer cells and validate them as therapeutic targets. METHODS: Transcriptome microarray and analysis using PANTHER pathway and StemChecker were performed to identify the most significantly expressed genes in tamoxifen-resistant and adriamycin-resistant MCF-7 breast cancer cells. Clinical relevance of the key genes was determined using Kaplan-Meier survival analyses on The Cancer Genome Atlas dataset of breast cancer patients. Gene overexpression/knockdown, spheroid formation, flow cytometric analysis, chromatin immunoprecipitation, immunocytochemistry, wound healing/transwell migration assays, and cancer stem cell transcription factor activation profiling array were used to elucidate the regulatory mechanism of integrin α11 expression. Tumour-bearing xenograft models were used to demonstrate integrin α11 is a potential therapeutic target. RESULTS: Integrin α11 was consistently upregulated in drug-resistant breast cancer cells, and its silencing inhibited cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT) while restoring sensitivity to anticancer drugs. HIF1α, GLI-1, and EZH2 contributed the most to the regulation of integrin α11 and EZH2 expression, with EZH2 being more necessary for EZH2 autoinduction than HIF1α and GLI-1. Additionally, unlike HIF1α or EZH2, GLI-1 was the sole transcription factor activated by integrin-linked focal adhesion kinase, indicating GLI-1 as a key driver of the EZH2-integrin α11 axis operating for cancer stem cell survival and EMT. Kaplan-Meier survival analysis using The Cancer Genome Atlas (TCGA) dataset also revealed both EZH2 and integrin α11 could be strong prognostic factors of relapse-free and overall survival in breast cancer patients. However, the superior efficacy of integrin α11 siRNA therapy over EZH2 siRNA treatment was demonstrated by enhanced inhibition of tumour growth and prolonged survival in murine models bearing tumours. CONCLUSION: Our findings elucidate that integrin α11 is upregulated by EZH2, forming a positive feedback circuit involving FAK-GLI-1 and contributing to drug resistance, cancer stem cell survival and EMT. Taken together, the results suggest integrin α11 as a promising prognostic marker and a powerful therapeutic target for drug-resistant breast cancer.
Assuntos
Neoplasias da Mama , Resistencia a Medicamentos Antineoplásicos , Proteína Potenciadora do Homólogo 2 de Zeste , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Células-Tronco Neoplásicas , RNA Interferente Pequeno , Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/mortalidade , Neoplasias da Mama/terapia , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Animais , Camundongos , Transição Epitelial-Mesenquimal/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , RNA Interferente Pequeno/genética , Linhagem Celular Tumoral , Progressão da Doença , Células MCF-7 , Proliferação de Células , Perfilação da Expressão GênicaRESUMO
GLI1, a key transcription factor of the Hedgehog (Hh) signaling pathway, plays an important role in the development of cancer. However, the function and mechanisms by which GLI1 regulates gene transcription are not fully understood in gastric cancer (GC). Here, we found that GLI1 induced the proliferation and metastasis of GC cells, accompanied by transcriptional upregulation of INHBA. This increased INHBA expression exerted a promoting activity on Smads signaling and then transcriptionally activated GLI1 expression. Notably, our results demonstrate that disrupting the interaction between GLI1 and INHBA could inhibit GC tumorigenesis in vivo. More intriguingly, we confirmed the N6-methyladenosine (m6A) activation mechanism of the Helicobacter pylori/FTO/YTHDF2/GLI1 pathway in GC cells. In conclusion, our study confirmed that the GLI1/INHBA positive feedback loop influences GC progression and revealed the mechanism by which H. pylori upregulates GLI1 expression through m6A modification. This positive GLI1/INHBA feedback loop suggests a novel noncanonical mechanism of GLI1 activity in GC and provides potential therapeutic targets for GC treatment.
Assuntos
Proliferação de Células , Progressão da Doença , Retroalimentação Fisiológica , Regulação Neoplásica da Expressão Gênica , Helicobacter pylori , Neoplasias Gástricas , Proteína GLI1 em Dedos de Zinco , Neoplasias Gástricas/patologia , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/genética , Proteína GLI1 em Dedos de Zinco/metabolismo , Proteína GLI1 em Dedos de Zinco/genética , Humanos , Animais , Linhagem Celular Tumoral , Camundongos , Transdução de Sinais , Infecções por Helicobacter/metabolismo , Infecções por Helicobacter/complicações , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Regulação para Cima , Masculino , Carcinogênese/genéticaRESUMO
Harnessing the developmental events of mesenchymal condensation to direct postnatal dental stem cell aggregation represents a cutting-edge and promising approach to tooth regeneration. Tooth avulsion is among the most prevalent and serious dental injuries, and odontogenic aggregates assembled by stem cells from human exfoliated deciduous teeth (SHED) have proven effective in revitalizing avulsed teeth after replantation in the clinical trial. However, whether and how SHED aggregates (SA) communicate with recipient components and promote synergistic tissue regeneration to support replanted teeth remains elusive. Here, it is shown that SA-mediated avulsed tooth regeneration involves periodontal restoration and recovery of recipient Gli1+ stem cells, which are mobilized and necessarily contribute to the reestablishment of the tooth-periodontal ligament-bone interface. Mechanistically, the release of extracellular vesicles (EVs) is revealed indispensable for the implanted SA to mobilize recipient Gli1+ cells and regenerate avulsed teeth. Furthermore, SHED aggregates-released EVs (SA-EVs) are featured with odontogenic properties linked to tissue regeneration, which enhance migration, proliferation, and differentiation of Gli1+ cells. Importantly, local application of SA-EVs per se empowers recipient Gli1+ cells and safeguards regeneration of avulsed teeth. Collectively, the findings establish a paradigm in which odontogenesis-featured EVs govern donor-recipient stem cell interplay to achieve tooth regeneration, inspiring cell-free translational regenerative strategies.
RESUMO
Mesenchymal tumors with GLI1 fusions or amplifications have recently emerged as a distinctive group of neoplasms. The terms GLI1-altered mesenchymal tumor or GLI1-altered soft tissue tumor serve as a nosological category, although the exact boundaries/criteria require further elucidation. We examined 16 tumors affecting predominantly adults (median age: 40 years), without sex predilection. Several patients had tumors of longstanding duration (>10 years). The most common primary site was soft tissue (n = 9); other sites included epidural tissue (n = 1), vertebra (n = 1), tongue (n = 1), hard palate (n = 1), and liver (n = 1). Histologically, the tumors demonstrated multinodular growth of cytologically uniform, ovoid-to-epithelioid, occasionally short spindled cells with delicate intratumoral vasculature and frequent myxoid stroma. Mitotic activity ranged from 0 to 8 mitoses/2 mm2 (mean 2). Lymphovascular invasion/protrusion of tumor cells into endothelial-lined vascular spaces was present or suspected in 6 cases. Necrosis, significant nuclear pleomorphism, or well-developed, fascicular spindle-cell growth were absent. Half demonstrated features of the newly proposed subset, "distinctive nested glomoid neoplasm." Tumors were consistently positive for CD56 (n = 5/5). A subset was stained with S100 protein (n = 7/13), SMA (n = 6/13), keratin (n = 2/9), EMA (n = 3/7), and CD99 (n = 2/6). Tumors harbored ACTB::GLI1 (n = 15) or PTCH1::GLI1 (n = 1) fusions. The assays used did not capture cases defined by GLI1 amplification. We also identified recurrent cytogenetic gains (1q, 5, 7, 8, 12, 12q13.2-ter, 21, and X). For patients with available clinical follow-up (n = 8), half were disease free. Half demonstrated distant metastases (lungs, bone, or soft tissue). Of cases without follow-up (n = 8), 2 were known recurrences, and 1 was presumed metastasis. Our results imply a more aggressive biological potential than currently reported. Given the possibility for metastasis and disease progression, even in cytologically bland, nested tumors, close clinical surveillance, akin to that for sarcoma management, may be indicated. The term GLI1-altered mesenchymal tumor with malignant potential is proposed.
Assuntos
Neoplasias de Tecido Conjuntivo e de Tecidos Moles , Sarcoma , Neoplasias de Tecidos Moles , Adulto , Humanos , Proteína GLI1 em Dedos de Zinco/genética , Neoplasias de Tecidos Moles/genética , Neoplasias de Tecidos Moles/patologia , Proteínas S100 , Sarcoma/patologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/análiseRESUMO
GLI1(12q13.3) amplification is identified in a subset of mesenchymal neoplasms with a distinct nested round cell/epithelioid phenotype. MDM2 and CDK4 genes are situated along the oncogenic 12q13-15 segment, amplification of which defines well-differentiated liposarcoma (WDLPS)/dedifferentiated liposarcoma (DDLPS). The 12q amplicon can occasionally include GLI1, a gene in close proximity to CDK4. We hereby describe the first cohort of GLI1/MDM2/CDK4 coamplified WD/DDLPS. The departmental database was queried retrospectively for all cases of WD/DDLPS having undergone next-generation (MSK-IMPACT) sequencing with confirmed MDM2, CDK4, and GLI1 coamplification. Clinicopathologic data was obtained from a review of the medical chart and available histologic material. Four hundred eighty-six WD/DDLPS cases underwent DNA sequencing, 92 (19%) of which harbored amplification of the GLI1 locus in addition to that of MDM2 and CDK4. These included primary tumors (n = 60), local recurrences (n = 29), and metastases (n = 3). Primary tumors were most frequently retroperitoneal (47/60, 78%), mediastinal (4/60, 7%), and paratesticular (3/60, 5%). Average age was 63 years, with a male:female ratio of 3:2. The cohort was comprised of DDLPS (86/92 [93%], 6 of which were WDLPS with early dedifferentiation) and WDLPS without any longitudinal evidence of dedifferentiation (6/92, 7%). One-fifth (13/86, 17%) of DDLPS cases showed no evidence of a well-differentiated component in any of the primary, recurrent, or metastatic specimens. Dedifferentiated areas mostly showed high-grade undifferentiated pleomorphic sarcoma-like (26/86,30%) and high-grade myxofibrosarcoma-like (13/86,16%) morphologies. A disproportionately increased incidence of meningothelial whorls with/without osseous metaplasia was observed as the predominant pattern in 16/86 (19%) cases, and GLI1-altered morphology as described was identified in a total of 10/86 (12%) tumors. JUN (1p32.1), also implicated in the pathogenesis of WD/DDLPS, was coamplified with all 3 of MDM2, CDK4, and GLI1 in 7/91 (8%) cases. Additional loci along chromosomal arms 1p and 6q, including TNFAIP3, LATS1, and ESR1, were also amplified in a subset of cases. In this large-scale cohort of GLI1 coamplified WD/DDLPS, we elucidate uniquely recurrent features including meningothelial whorl-like and GLI-altered morphology in dedifferentiated areas. Assessment of tumor location (retroperitoneal or mediastinal), identification of a well-differentiated liposarcoma component, and coamplification of other spatially discrete genomic segments (1p and 6q) might aid in distinction from tumors with true driver GLI1 alterations.
Assuntos
Quinase 4 Dependente de Ciclina , Amplificação de Genes , Lipossarcoma , Proteína GLI1 em Dedos de Zinco , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Biomarcadores Tumorais/genética , Quinase 4 Dependente de Ciclina/genética , Lipossarcoma/genética , Lipossarcoma/patologia , Proteínas Proto-Oncogênicas c-mdm2/genética , Estudos Retrospectivos , Proteína GLI1 em Dedos de Zinco/genéticaRESUMO
BACKGROUND: Sonic Hedgehog (SHH) is a fundamental signaling pathway that controls tissue reconstruction, stem cell biology, and differentiation and has a role in gut tissue homeostasis and development. Dysregulation of SHH leads to the development of HCC. METHODS, AND RESULTS: The present study was conducted to compare the effects of mesenchymal stem cells (MSCs) and curcumin on SHH molecular targets in an experimental model of HCC in rats. One hundred rats were divided equally into the following groups: control group, HCC group, HCC group received MSCs, HCC group received curcumin, and HCC group received MSCs and curcumin. Histopathological examinations were performed, and gene expression of SHH signaling target genes (SHH, PTCH1, SMOH, and GLI1) was assessed by real-time PCR in rat liver tissue. Results showed that SHH target genes were significantly upregulated in HCC-untreated rat groups and in MSC-treated groups, with no significant difference between them. Administration of curcumin with or without combined administration of MSCs led to a significant down-regulation of SHH target genes, with no significant differences between both groups. As regards the histopathological examination of liver tissues, both curcumin and MSCs, either through separate use or their combined use, led to a significant restoration of normal liver pathology. CONCLUSIONS: In conclusion, SHH signaling is upregulated in the HCC experimental model. MSCs do not inhibit the upregulated SHH target genes in HCC. Curcumin use with or without MSCs administration led to a significant down-regulation of SHH signaling in HCC and a significant restoration of normal liver pathology.
Assuntos
Carcinoma Hepatocelular , Curcumina , Proteínas Hedgehog , Neoplasias Hepáticas , Células-Tronco Mesenquimais , Transdução de Sinais , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Animais , Curcumina/farmacologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Transdução de Sinais/efeitos dos fármacos , Ratos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Transplante de Células-Tronco Mesenquimais/métodos , Masculino , Modelos Animais de Doenças , Receptor Patched-1/genética , Receptor Patched-1/metabolismo , Proteína GLI1 em Dedos de Zinco/metabolismo , Proteína GLI1 em Dedos de Zinco/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Fígado/efeitos dos fármacosRESUMO
Aberrant activation of the Hedgehog (Hh) signaling pathway, through which the GLI family of transcription factors (TF) is stimulated, is commonly observed in cancer cells. One well-established mechanism of this increased activity is through the inactivation of Suppressor of Fused (SUFU), a negative regulator of the Hh pathway. Relief from negative regulation by SUFU facilitates GLI activity and induction of target gene expression. Here, we demonstrate a novel role for SUFU as a promoter of GLI activity in pancreatic ductal adenocarcinoma (PDAC). In non-ciliated PDAC cells unresponsive to Smoothened agonism, SUFU overexpression increases GLI transcriptional activity. Conversely, knockdown (KD) of SUFU reduces the activity of GLI in PDAC cells. Through array PCR analysis of GLI target genes, we identified B-cell lymphoma 2 (BCL2) among the top candidates down-regulated by SUFU KD. We demonstrate that SUFU KD results in reduced PDAC cell viability, and overexpression of BCL2 partially rescues the effect of reduced cell viability by SUFU KD. Further analysis using as a model GLI1, a major TF activator of the GLI family in PDAC cells, shows the interaction of SUFU and GLI1 in the nucleus through previously characterized domains. Chromatin immunoprecipitation (ChIP) assay shows the binding of both SUFU and GLI1 at the promoter of BCL2 in PDAC cells. Finally, we demonstrate that SUFU promotes GLI1 activity without affecting its protein stability. Through our findings, we propose a novel role of SUFU as a positive regulator of GLI1 in PDAC, adding a new mechanism of Hh/GLI signaling pathway regulation in cancer cells.
Assuntos
Neoplasias Pancreáticas , Proteínas Repressoras , Humanos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteína GLI1 em Dedos de Zinco/genética , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas c-bcl-2 , Neoplasias PancreáticasRESUMO
Carcinoma-associated fibroblasts (CAFs) play an important role in the progression of multiple malignancies. Secretion of cytokines and growth factors underlies the pro-tumoral effect of CAFs. Although this paracrine function has been extensively documented, the molecular mechanisms controlling the expression of these factors remain elusive. In this study, we provide evidence of a novel CAF transcriptional axis regulating the expression of SDF1, a major driver of cancer cell migration, involving the transcription factor GLI1 and histone acetyltransferase p300. We demonstrate that conditioned media from CAFs overexpressing GLI1 induce the migration of pancreatic cancer cells, and this effect is impaired by an SDF1-neutralizing antibody. Using a combination of co-immunoprecipitation, proximity ligation assay and chromatin immunoprecipitation assay, we further demonstrate that GLI1 and p300 physically interact in CAFs to co-occupy and drive SDF1 promoter activity. Mapping experiments highlight the requirement of GLI1 N-terminal for the interaction with p300. Importantly, knockdowns of both GLI1 and p300 reduce SDF1 expression. Further analysis shows that knockdown of GLI1 decreases SDF1 promoter activity, p300 recruitment, and levels of its associated histone marks (H4ac, H3K27ac, and H3K14ac). Finally, we show that the integrity of two GLI binding sites in the SDF1 promoter is required for p300 recruitment. Our findings define a new role for the p300-GLI1 complex in the regulation of SDF1, providing new mechanistic insight into the molecular events controlling pancreatic cancer cells migration.
Assuntos
Fibroblastos Associados a Câncer , Neoplasias Pancreáticas , Humanos , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Movimento Celular , Imunoprecipitação da Cromatina , Neoplasias Pancreáticas/patologia , Transdução de Sinais , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismo , Quimiocina CXCL12/metabolismo , Neoplasias PancreáticasRESUMO
Synovial sarcoma (SS) is a rare soft-tissue tumor characterized by a monomorphic blue spindle cell histology and variable epithelial differentiation. Morphologically, SSs may be confused with other sarcomas. Systemic treatment is more effective for patients with high-risk SSs, patients with advanced disease, and younger patients. However, further studies are required to find new prognostic biomarkers. Herein, we describe the morphological, molecular, and clinical findings, using a wide immunohistochemical panel, of a series of SS cases. We studied 52 cases confirmed as SSs by morphological diagnosis and/or molecular studies. Clinical data (gender, age, tumor size, tumor location, resection margins, adjuvant treatment, recurrences, metastasis, and survival) were also retrieved for each patient. All the available H&E slides were examined by four pathologists. Three tissue microarrays (TMAs) were constructed for each of the tumors, and a wide immunohistochemical panel was performed. For time-to-event variables, survival analysis was performed using Kaplan-Meier curves and log-rank testing, or Cox regression. Statistical significance was considered at p < 0.05. The mean age of our patients was 40.33, and the median was 40.5 years. We found a predominance of males versus females (1.7:1). The most frequent morphological subtype was monophasic. TRPS1, SS18-SSX, and SSX-C-terminus were positive in 96% of cases. GLI1 expression was strong in six and focal (cytoplasmic) in twenty patients. Moreover, BCOR was expressed in more than half of SSs. Positive expression of both proteins, BCOR and GLI1, was correlated with a worse prognosis. Multivariate analysis was also performed, but only BCOR expression appeared to be significant. The combination of GLI1 and BCOR antibodies can be used to group SSs into three risk groups (low, intermediate, and high risk). We hypothesize that these findings could identify which patients would benefit from receiving adjuvant treatment and which would not. Moreover, these markers could represent therapeutic targets in advanced stages. However, further, larger series of SSs and molecular studies are necessary to corroborate our present findings.
Assuntos
Biomarcadores Tumorais , Imuno-Histoquímica , Proteínas Proto-Oncogênicas , Proteínas Repressoras , Sarcoma Sinovial , Proteína GLI1 em Dedos de Zinco , Humanos , Sarcoma Sinovial/metabolismo , Sarcoma Sinovial/patologia , Sarcoma Sinovial/genética , Masculino , Feminino , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Adulto , Pessoa de Meia-Idade , Prognóstico , Biomarcadores Tumorais/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteína GLI1 em Dedos de Zinco/metabolismo , Proteína GLI1 em Dedos de Zinco/genética , Idoso , Adulto Jovem , Adolescente , Estimativa de Kaplan-MeierRESUMO
For a wide range of chronic autoimmune and inflammatory diseases in both adults and children, synthetic glucocorticoids (GCs) are one of the most effective treatments. However, besides other adverse effects, GCs inhibit bone mass at multiple levels, and at different ages, especially in puberty. Although extensive studies have investigated the mechanism of GC-induced osteoporosis, their target cell populations still be obscure. Here, our data show that the osteoblast subpopulation among Gli1+ metaphyseal mesenchymal progenitors (MMPs) is responsive to GCs as indicated by lineage tracing and single-cell RNA sequencing experiments. Furthermore, the proliferation and differentiation of Gli1+ MMPs are both decreased, which may be because GCs impair the oxidative phosphorylation(OXPHOS) and aerobic glycolysis of Gli1+ MMPs. Teriparatide, as one of the potential treatments for GCs in bone mass, is sought to increase bone volume by increasing the proliferation and differentiation of Gli1+ MMPs in vivo. Notably, our data demonstrate teriparatide ameliorates GC-caused bone defects by targeting Gli1+ MMPs. Thus, Gli1+ MMPs will be the potential mesenchymal progenitors in response to diverse pharmaceutical administrations in regulating bone formation.
Assuntos
Glucocorticoides , Células-Tronco Mesenquimais , Osteoporose , Animais , Camundongos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Glucocorticoides/efeitos adversos , Glucocorticoides/farmacologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Osteoporose/induzido quimicamente , Osteoporose/metabolismo , Osteoporose/patologia , Teriparatida/farmacologia , Proteína GLI1 em Dedos de Zinco/metabolismo , Proteína GLI1 em Dedos de Zinco/genéticaRESUMO
Aberrant activation of hedgehog (Hh) signaling has been implicated in various cancers. Current FDA-approved inhibitors target the seven-transmembrane receptor Smoothened, but resistance to these drugs has been observed. It has been proposed that a more promising strategy to target this pathway is at the GLI1 transcription factor level. GANT61 was the first small molecule identified to directly suppress GLI-mediated activity; however, its development as a potential anti-cancer agent has been hindered by its modest activity and aqueous chemical instability. Our study aimed to identify novel GLI1 inhibitors. JChem searches identified fifty-two compounds similar to GANT61 and its active metabolite, GANT61-D. We combined high-throughput cell-based assays and molecular docking to evaluate these analogs. Five of the fifty-two GANT61 analogs inhibited activity in Hh-responsive C3H10T1/2 and Gli-reporter NIH3T3 cellular assays without cytotoxicity. Two of the GANT61 analogs, BAS 07019774 and Z27610715, reduced Gli1 mRNA expression in C3H10T1/2 cells. Treatment with BAS 07019774 significantly reduced cell viability in Hh-dependent glioblastoma and lung cancer cell lines. Molecular docking indicated that BAS 07019774 is predicted to bind to the ZF4 region of GLI1, potentially interfering with its ability to bind DNA. Our findings show promise in developing more effective and potent GLI inhibitors.
Assuntos
Proteínas Hedgehog , Simulação de Acoplamento Molecular , Piridinas , Pirimidinas , Proteína GLI1 em Dedos de Zinco , Piridinas/farmacologia , Piridinas/química , Proteína GLI1 em Dedos de Zinco/metabolismo , Proteína GLI1 em Dedos de Zinco/genética , Pirimidinas/farmacologia , Pirimidinas/química , Proteínas Hedgehog/metabolismo , Humanos , Animais , Camundongos , Linhagem Celular Tumoral , Células NIH 3T3 , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Transdução de Sinais/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacosRESUMO
GLI1-altered mesenchymal tumors comprise a group of seemingly unrelated entities, including pericytoma with t(7;12) translocation, plexiform fibromyxoma, gastroblastoma, malignant epithelioid neoplasm with GLI1 rearrangements, and GLI1-amplified mesenchymal neoplasms. Herein, we report a high-grade uterine sarcoma harboring a novel PAMR1::GLI1 fusion and present a literature review of GLI1-altered mesenchymal neoplasms of the gynecologic tract. A 57-year-old female presented with an abdomino-pelvic mass, felt since a decade prior. Magnetic resonance imaging showed a heterogenous myometrial mass extending beyond the serosa. The patient underwent oncologic surgical resection. Gross examination revealed a perforated multi-nodular uterine tumor (21 cm) with a firm white and soft fleshy cut surface, featuring hemorrhage and necrosis. The tumor was morphologically heterogenous, disclosing frankly sarcomatous areas composed of pleomorphic spindle and focally epithelioid cells, intermingled with a component of low-grade spindle cells arranged in fascicles. There was a rich vascular network and zones of necrosis with peripheral amianthoid-like collagen plaques. Lymphovascular invasion and metastasis to lymph nodes and omentum were present. The tumor was immunopositive for CD10 and cyclinD1, and negative for cytokeratins, myogenic, melanotic, and hormonal markers. ArcherTM Fusion Sarcoma Assay detected PAMR1(exon1)::GLI1(exon4) fusion, confirmed on RT-PCR and Sanger sequencing. The patient received chemo-radiotherapy, however, developed metastatic recurrence and demised 18 months post-surgery. Altogether, this is a rare and diagnostically challenging case of a uterine sarcoma harboring a novel GLI1 fusion. Emerging GLI/Hedgehog inhibitors provide clinical relevance to recognizing these tumors in modern pathology.
Assuntos
Proteínas Hedgehog , Sarcoma , Feminino , Humanos , Pessoa de Meia-Idade , Sarcoma/genética , Necrose , Proteína GLI1 em Dedos de Zinco/genéticaRESUMO
Meningioma is a common brain tumour which has neither a specific detection nor treatment method. The Sonic hedgehog (Shh) cell signaling pathway is a crucial regulatory pathway of mammalian organogenesis and tumorigenesis including meningioma. Shh cell signalling pathway cascade function by main transcription factor Gli1 and which further regulates in its downstream to Pax6 and Nkx2.2. This current study is aimed to explore the regulation of the Sonic hedgehog-Gli1 cell signaling pathway and its potential downstream targets in meningioma samples. A total of 24 surgically resected meningioma samples were used in this current study.Cytological changes were assessed using electron microscopic techniques as well as hematoxylin & eosin and DAPI staining. The expression pattern of Gli1, Nkx2.2 and Pax6 transcription factors were determined by using immunohistochemistry. The mRNA expression was assessed using RT-qPCR assays. Later, the whole transcriptome analysis of samples was performed with the amploseq technique. Results were compared with those obtained in normal human brain tissue (or normal meninges). Compared to the normal human brain tissue, meningioma samples showed crowded nuclei with morphological changes. Transcription factor Nkx2.2 expressed highly in all samples (24/24, 100%). Twenty-one of the 24 meningiomas (88%) showed high Gli1 and Pax6 expression. Whole transcriptome analysis of two meningioma samples also exhibited a very high increase in Gli1 expression signal in meningioma samples as compare to normal control. Hence, we may conclude that the Shh-Gli1 pathway is aberrantly activated in meningioma cells and is canonically upregulating the expression of transcription factors Pax6 and Nkx2.2. Supplementary Information: The online version contains supplementary material available at 10.1007/s12291-022-01085-1.
RESUMO
Peripheral nerves are organized into discrete compartments. Axons, Schwann cells (SCs), and endoneurial fibroblasts (EFs) reside within the endoneurium and are surrounded by the perineurium, a cellular sheath comprised of layers of perineurial glia (PNG). SC secretion of Desert Hedgehog (Dhh) regulates this organization. In Dhh nulls, the perineurium is deficient and the endoneurium is subdivided into small compartments termed minifascicles. Human Dhh mutations cause a neuropathy with similar defects. Here we examine the role of Gli1, a canonical transcriptional effector of hedgehog signaling, in regulating peripheral nerve organization in mice of both genders. We identify PNG, EFs, and pericytes as Gli1-expressing cells by genetic fate mapping. Although expression of Dhh by SCs and Gli1 in target cells is coordinately regulated with myelination, Gli1 expression unexpectedly persists in Dhh null EFs. Thus, Gli1 is expressed in EFs noncanonically (i.e., independent of hedgehog signaling). Gli1 and Dhh also have nonredundant activities. Unlike Dhh nulls, Gli1 nulls have a normal perineurium. Like Dhh nulls, Gli1 nulls form minifascicles, which we show likely arise from EFs. Thus, Dhh and Gli1 are independent signals: Gli1 is dispensable for perineurial development but functions cooperatively with Dhh to drive normal endoneurial development. During development, Gli1 also regulates endoneurial extracellular matrix production, nerve vascular organization, and has modest, nonautonomous effects on SC sorting and myelination of axons. Finally, in adult nerves, induced deletion of Gli1 is sufficient to drive minifascicle formation. Thus, Gli1 regulates the development and is required to maintain the endoneurial architecture of peripheral nerves.SIGNIFICANCE STATEMENT Peripheral nerves are organized into distinct cellular/ECM compartments: the epineurium, perineurium, and endoneurium. This organization, with its associated cellular constituents, is critical for the structural and metabolic support of nerves and their response to injury. Here, we show that Gli1, a transcription factor normally expressed downstream of hedgehog signaling, is required for the proper organization of the endoneurium but not the perineurium. Unexpectedly, Gli1 expression by endoneurial cells is independent of, and functions nonredundantly with, Schwann Cell-derived Desert Hedgehog in regulating peripheral nerve architecture. These results further delineate how peripheral nerves acquire their distinctive organization during normal development, and highlight mechanisms that may regulate their reorganization in pathologic settings, including peripheral neuropathies and nerve injury.
Assuntos
Nervos Periféricos/metabolismo , Proteína GLI1 em Dedos de Zinco/metabolismo , Animais , Axônios/metabolismo , Proteína 2 de Resposta de Crescimento Precoce/genética , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Feminino , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Knockout , Células de Schwann/metabolismo , Proteína GLI1 em Dedos de Zinco/genéticaRESUMO
Gastric cancer (GC) is a highly prevalent and aggressive malignancy with a poor prognosis. Recent evidence suggested that metallothionein 1 M (MT1M) may play a critical role in cancer development, progression, and drug resistance; however, its role in GC remains largely unknown. In this study, we investigated the expression and function of MT1M in GC both in vitro and in vivo. We found that MT1M expression was significantly downregulated in GC tissues and cell lines. Decreased expression of MT1M was associated with worse clinical prognosis, particularly in patients treated with 5-fluorouracil. Low expression of MT1M was indicative of poor overall survival (OS, HR 0.56 [95% CI 0.37-0.84], P < 0.005), first progression survival (FP, HR 0.54 [95% CI 0.36-0.79], P < 0.005), and post-progression survival (PPS, HR 0.65 [95% CI 0.45-0.94], P < 0.05). We also demonstrated that overexpression of MT1M inhibited cell proliferation and induced apoptosis in GC cells and in tumor xenografts, and it improved chemosensitivity to 5-fluorouracil. Furthermore, we found that MT1M overexpression could inhibit stem cell characteristics by targeting GLI1 and affecting GLI1 ubiquitination. Collectively, these findings indicated that MT1M may act as a tumor suppressor in GC and could serve as a potential therapeutic target to attenuate stemness and chemotherapy resistance of GC.
Assuntos
Neoplasias Gástricas , Humanos , Neoplasias Gástricas/metabolismo , Proteínas Hedgehog/metabolismo , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismo , Linhagem Celular Tumoral , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Metalotioneína/genéticaRESUMO
The proliferation of spermatogonia directly affects spermatogenesis and male fertility, but its underlying molecular mechanisms are poorly understood. In this study, Smoothened (Smo), the central transducer of Hedgehog signaling pathway, was characterized in medaka (Oryzias latipes), and its role and underlying mechanisms in the proliferation of spermatogonia were investigated. Smo was highly expressed in spermatogonia. In ex vivo testicular organ culture and a spermatogonial cell line (SG3) derived from medaka mature testis, Smo activation promoted spermatogonia proliferation, while its inhibition induced apoptosis. The expression of glioma-associated oncogene homolog 1 (gli1) and regulator of cell cycle (rgcc) was significantly upregulated in SG3 after Smo activation. Furthermore, Gli1 transcriptionally upregulated the expression of rgcc, and Rgcc overexpression rescued cell apoptosis caused by Smo or Gli1 inhibition. Co-immunoprecipitation assay indicated that Rgcc could interact with cyclin-dependent kinase 1 (Cdk1) to regulate the cell cycle of spermatogonia. Collectively, our study firstly reveals that Smo mediates the proliferation of spermatogonia through Gli1-Rgcc-Cdk1 axis. In addition, Smo and Gli1 are necessary of the survival of spermatogonia. This study deepens our understanding of spermatogonia proliferation and survival at the molecular level, and provides insights into male fertility control and reproductive disease treatment.
Assuntos
Oryzias , Animais , Masculino , Espermatogônias/metabolismo , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismo , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Proliferação de Células , Proteínas Hedgehog/metabolismoRESUMO
Renal cell carcinoma (RCC) is the second commonest urological malignant neoplasm and mortality rate of patients with RCC appears to be increasing each year. Thus, further understanding of the molecular mechanisms responsible for the development and progression of RCC is of particular importance. Here, we report that atypical chemokine receptor 3 (ACKR3) orchestrates the Hedgehog (Hh)-GLI1 signaling to promote RCC progression. The expression of ACKR3 is elevated in RCC tissues, which is associated with malignant and clinical outcomes of RCC, and ACKR3 expression is positively correlated with GLI1 expression in RCC tissues. Mechanically, Hh promotes RCC progression through GLI1-mediated ACKR3 transcription by the directly binding of GLI1 to ACKR3 gene, while CXCL12-ACKR3 axis simultaneously enhances Hh activation via the binding of ACKR3 to Smoothened (SMO), a receptor in Hh pathway, resulting in the upregulation of SMO phosphorylation that potentiates downstream signal activity and consequently contributes to RCC progression. Thus, our findings may provide with the evidence of developing a novel treatment method with specific target for RCC.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Humanos , Carcinoma de Células Renais/genética , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteína GLI1 em Dedos de Zinco/genética , Fatores de Transcrição/genética , Neoplasias Renais/genética , Neoplasias Renais/patologiaRESUMO
The Hedgehog pathway gene Gli1 has been proposed to mark a subpopulation of skeletal stem cells (SSCs) in craniofacial bone. Skeletal stem cells (SSCs) are multi-potent cells crucial for the development and homeostasis of bone. Recent studies on long bones have suggested that skeletal stem cells in endochondral or intramembranous ossification sites have different differentiation capacities. However, this has not been well-defined in neural crest derived bones. Generally, the long bones are derived from mesoderm and follow an endochondral ossification model, while most of the cranial bones are neural crest (NC) in origin and follow an intramembranous ossification model. The mandible is unique: It is derived from the neural crest lineage but makes use of both modes of ossification. Early in fetal development, the mandibular body is generated by intramembranous ossification with subsequent endochondral ossification forming the condyle. The identities and properties for SSCs in these two sites remain unknown. Here, we use genetic lineage tracing in mouse to identify cells expressing the Hedgehog responsive gene Gli1, which is thought to mark the tissue resident SSCs. We track the Gli1+ cells, comparing cells within the perichondrium to those in the periosteum covering the mandibular body. In juvenile mice, these have distinct differentiation and proliferative potential. We also assess the presence of Sox10+ cells, thought to mark neural crest stem cells, but find no substantial population associated with the mandibular skeleton, suggesting that Sox10+ cells have limited contribution to maintaining postnatal mandibular bone. All together, our study indicates that the Gli1+ cells display distinct and limited differentiation capacity dependent on their regional associations.