GLI1+ Cells Contribute to Vascular Remodeling in Pulmonary Hypertension.

BACKGROUND: The precise origin of newly formed ACTA2+ (alpha smooth muscle actin-positive) cells appearing in nonmuscularized vessels in the context of pulmonary hypertension is still debatable although it is believed that they predominantly derive from preexisting vascular smooth muscle cells (VSMCs). METHODS: Gli1Cre-ERT2; tdTomatoflox mice were used to lineage trace GLI1+ (glioma-associated oncogene homolog 1-positive) cells in the context of pulmonary hypertension using 2 independent models of vascular remodeling and reverse remodeling: hypoxia and cigarette smoke exposure. Hemodynamic measurements, right ventricular hypertrophy assessment, flow cytometry, and histological analysis of thick lung sections followed by state-of-the-art 3-dimensional reconstruction and quantification using Imaris software were used to investigate the contribution of GLI1+ cells to neomuscularization of the pulmonary vasculature. RESULTS: The data show that GLI1+ cells are abundant around distal, nonmuscularized vessels during steady state, and this lineage contributes to around 50% of newly formed ACTA2+ cells around these normally nonmuscularized vessels. During reverse remodeling, cells derived from the GLI1+ lineage are largely cleared in parallel to the reversal of muscularization. Partial ablation of GLI1+ cells greatly prevented vascular remodeling in response to hypoxia and attenuated the increase in right ventricular systolic pressure and right heart hypertrophy. Single-cell RNA sequencing on sorted lineage-labeled GLI1+ cells revealed an Acta2high fraction of cells with pathways in cancer and MAPK (mitogen-activated protein kinase) signaling as potential players in reprogramming these cells during vascular remodeling. Analysis of human lung-derived material suggests that GLI1 signaling is overactivated in both group 1 and group 3 pulmonary hypertension and can promote proliferation and myogenic differentiation. CONCLUSIONS: Our data highlight GLI1+ cells as an alternative cellular source of VSMCs in pulmonary hypertension and suggest that these cells and the associated signaling pathways represent an important therapeutic target for further studies.

Gli1 labels progenitors during chondrogenesis in postnatal mice.

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.

CPLANE protein INTU regulates growth and patterning of the mouse lungs through cilia-dependent Hh signaling.

Congenital lung malformations are fatal at birth in their severe forms. Prevention and early intervention of these birth defects require a comprehensive understanding of the molecular mechanisms of lung development. We find that the loss of inturned (Intu), a cilia and planar polarity effector gene, severely disrupts growth and branching morphogenesis of the mouse embryonic lungs. Consistent with our previous results indicating an important role for Intu in ciliogenesis and hedgehog (Hh) signaling, we find greatly reduced number of primary cilia in both the epithelial and mesenchymal tissues of the lungs. We also find significantly reduced expression of Gli1 and Ptch1, direct targets of Hh signaling, suggesting disruption of cilia-dependent Hh signaling in Intu mutant lungs. An agonist of the Hh pathway activator, smoothened, increases Hh target gene expression and tubulogenesis in explanted wild type, but not Intu mutant, lungs, suggesting impaired Hh signaling response underlying lung morphogenetic defects in Intu mutants. Furthermore, removing both Gli2 and Intu completely abolishes branching morphogenesis of the lung, strongly supporting a mechanism by which Intu regulates lung growth and patterning through cilia-dependent Hh signaling. Moreover, a transcriptomics analysis identifies around 200 differentially expressed genes (DEGs) in Intu mutant lungs, including known Hh target genes Gli1, Ptch1/2 and Hhip. Genes involved in muscle differentiation and function are highly enriched among the DEGs, consistent with an important role of Hh signaling in airway smooth muscle differentiation. In addition, we find that the difference in gene expression between the left and right lungs diminishes in Intu mutants, suggesting an important role of Intu in asymmetrical growth and patterning of the mouse lungs.

Targeting cancer hallmark vulnerabilities in hematologic malignancies by interfering with Hedgehog/GLI signaling.

Understanding the genetic alterations, disrupted signaling pathways, and hijacked mechanisms in oncogene-transformed hematologic cells is critical for the development of effective and durable treatment strategies against liquid tumors. In this review, we focus on the specific involvement of the Hedgehog (HH)/GLI pathway in the manifestation and initiation of various cancer features in hematologic malignancies, including multiple myeloma, T- and B-cell lymphomas, and lymphoid and myeloid leukemias. By reviewing canonical and noncanonical, Smoothened-independent HH/GLI signaling and summarizing preclinical in vitro and in vivo studies in hematologic malignancies, we elucidate common molecular mechanisms by which HH/GLI signaling controls key oncogenic processes and cancer hallmarks such as cell proliferation, cancer stem cell fate, genomic instability, microenvironment remodeling, and cell survival. We also summarize current clinical trials with HH inhibitors and discuss successes and challenges, as well as opportunities for future combined therapeutic approaches. By providing a bird's eye view of the role of HH/GLI signaling in liquid tumors, we suggest that a comprehensive understanding of the general oncogenic effects of HH/GLI signaling on the formation of cancer hallmarks is essential to identify critical vulnerabilities within tumor cells and their supporting remodeled microenvironment, paving the way for the development of novel and efficient personalized combination therapies for hematologic malignancies.

A role for TGFß signaling in Gli1+ tendon and enthesis cells.

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.

Combinatorial Gli activity directs immune infiltration and tumor growth in pancreatic cancer.

Proper Hedgehog (HH) signaling is essential for embryonic development, while aberrant HH signaling drives pediatric and adult cancers. HH signaling is frequently dysregulated in pancreatic cancer, yet its role remains controversial, with both tumor-promoting and tumor-restraining functions reported. Notably, the GLI family of HH transcription factors (GLI1, GLI2, GLI3), remain largely unexplored in pancreatic cancer. We therefore investigated the individual and combined contributions of GLI1-3 to pancreatic cancer progression. At pre-cancerous stages, fibroblast-specific Gli2/Gli3 deletion decreases immunosuppressive macrophage infiltration and promotes T cell infiltration. Strikingly, combined loss of Gli1/Gli2/Gli3 promotes macrophage infiltration, indicating that subtle changes in Gli expression differentially regulate immune infiltration. In invasive tumors, Gli2/Gli3 KO fibroblasts exclude immunosuppressive myeloid cells and suppress tumor growth by recruiting natural killer cells. Finally, we demonstrate that fibroblasts directly regulate macrophage and T cell migration through the expression of Gli-dependent cytokines. Thus, the coordinated activity of GLI1-3 directs the fibroinflammatory response throughout pancreatic cancer progression.

The BAF chromatin complex component SMARCC1 does not mediate GLI transcriptional repression of Hedgehog target genes in limb buds.

Transcriptional responses to the Hedgehog (HH) signaling pathway are primarily modulated by GLI repression in the mouse limb. Previous studies suggested a role for the BAF chromatin remodeling complex in mediating GLI repression. Consistent with this possibility, the core BAF complex protein SMARCC1 is present at most active limb enhancers including the majority of GLI enhancers. However, in contrast to GLI repression which reduces chromatin accessibility, SMARCC1 maintains chromatin accessibility at most enhancers, including those bound by GLI. Moreover, SMARCC1 binding at GLI-regulated enhancers occurs independently of GLI3. Consistent with previous studies, some individual GLI target genes are mis-regulated in Smarcc1 conditional knockouts, though most GLI target genes are unaffected. Moreover, SMARCC1 is not necessary for mediating constitutive GLI repression in HH mutant limb buds. We conclude that SMARCC1 does not mediate GLI3 repression, which we propose utilizes alternative chromatin remodeling complexes.

Conditioned Medium From Reactive Astrocytes Inhibits Proliferation, Resistance, and Migration of p53-Mutant Glioblastoma Spheroid Through GLI-1 Downregulation.

Glioblastoma (GBM) aggressiveness is partly driven by the reactivation of signaling pathways such as Sonic hedgehog (SHH) and the interaction with its microenvironment. SHH pathway activation is one of the phenomena behind the glial transformation in response to tumor growth. The reactivation of the SHH signaling cascade during GBM-astrocyte interaction is highly relevant to understanding the mechanisms used by the tumor to modulate the adjacent stroma. The role of reactive astrocytes considering SHH signaling during GBM progression is investigated using a 3D in vitro model. T98G GBM spheroids displayed significant downregulation of SHH (61.4 ± 9.3%), GLI-1 (6.5 ± 3.7%), Ki-67 (33.7 ± 8.1%), and mutant MTp53 (21.3 ± 10.6%) compared to the CONTROL group when incubated with conditioned medium of reactive astrocytes (CM-AST). The SHH pathway inhibitor, GANT-61, significantly reduced previous markers (SHH = 43.0 ± 12.1%; GLI-1 = 9.5 ± 3.4%; Ki-67 = 31.9 ± 4.6%; MTp53 = 6.5 ± 7.5%) compared to the CONTROL, and a synergistic effect could be observed between GANT-61 and CM-AST. The volume (2.0 ± 0.2 × 107 µm³), cell viability (80.4 ± 3.2%), and migration (41 ± 10%) of GBM spheroids were significantly reduced in the presence of GANT-61 and CM-AST when compared to CM-AST after 72 h (volume = 2.3 ± 0.4 × 107 µm³; viability = 92.2 ± 6.5%; migration = 102.5 ± 14.6%). Results demonstrated that factors released by reactive astrocytes promoted a neuroprotective effect preventing GBM progression using a 3D in vitro model potentiated by SHH pathway inhibition.

Positive GLI1/INHBA feedback loop drives tumor progression in gastric cancer.

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.

Ciliopathic micrognathia is caused by aberrant skeletal differentiation and remodeling.

Ciliopathies represent a growing class of diseases caused by defects in microtubule-based organelles called primary cilia. Approximately 30% of ciliopathies are characterized by craniofacial phenotypes such as craniosynostosis, cleft lip/palate and micrognathia. Patients with ciliopathic micrognathia experience a particular set of difficulties, including impaired feeding and breathing, and have extremely limited treatment options. To understand the cellular and molecular basis for ciliopathic micrognathia, we used the talpid2 (ta2 ), a bona fide avian model for the human ciliopathy oral-facial-digital syndrome subtype 14. Histological analyses revealed that the onset of ciliopathic micrognathia in ta2 embryos occurred at the earliest stages of mandibular development. Neural crest-derived skeletal progenitor cells were particularly sensitive to a ciliopathic insult, undergoing unchecked passage through the cell cycle and subsequent increased proliferation. Furthermore, whereas neural crest-derived skeletal differentiation was initiated, osteoblast maturation failed to progress to completion. Additional molecular analyses revealed that an imbalance in the ratio of bone deposition and resorption also contributed to ciliopathic micrognathia in ta2 embryos. Thus, our results suggest that ciliopathic micrognathia is a consequence of multiple aberrant cellular processes necessary for skeletal development, and provide potential avenues for future therapeutic treatments.

Stromal androgen and hedgehog signaling regulates stem cell niches in pubertal prostate development.

Stromal androgen-receptor (AR) action is essential for prostate development, morphogenesis and regeneration. However, mechanisms underlying how stromal AR maintains the cell niche in support of pubertal prostatic epithelial growth are unknown. Here, using advanced mouse genetic tools, we demonstrate that selective deletion of stromal AR expression in prepubescent Shh-responsive Gli1-expressing cells significantly impedes pubertal prostate epithelial growth and development. Single-cell transcriptomic analyses showed that AR loss in these prepubescent Gli1-expressing cells dysregulates androgen signaling-initiated stromal-epithelial paracrine interactions, leading to growth retardation of pubertal prostate epithelia and significant development defects. Specifically, AR loss elevates Shh-signaling activation in both prostatic stromal and adjacent epithelial cells, directly inhibiting prostatic epithelial growth. Single-cell trajectory analyses further identified aberrant differentiation fates of prostatic epithelial cells directly altered by stromal AR deletion. In vivo recombination of AR-deficient stromal Gli1-lineage cells with wild-type prostatic epithelial cells failed to develop normal prostatic epithelia. These data demonstrate previously unidentified mechanisms underlying how stromal AR-signaling facilitates Shh-mediated cell niches in pubertal prostatic epithelial growth and development.

GLI1-Altered Mesenchymal Tumors With ACTB or PTCH1 Fusion: A Molecular and Clinicopathologic Analysis.

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.

GLI1 Coamplification in Well-Differentiated/Dedifferentiated Liposarcomas: Clinicopathologic and Molecular Analysis of 92 Cases.

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.

Staphylococcal enterotoxin B exposed to pregnant rats inhibits the hedgehog signaling pathway in thymic T lymphocytes of the offspring.

The Hedgehog (Hh) signaling pathway is involved in T cell differentiation and development and plays a major regulatory part in different stages of T cell development. A previous study by us suggested that prenatal exposure to staphylococcal enterotoxin B (SEB) changed the percentages of T cell subpopulation in the offspring thymus. However, it is unclear whether prenatal SEB exposure impacts the Hh signaling pathway in thymic T cells. In the present study, pregnant rats at gestational day 16 were intravenously injected once with 15 µg SEB, and the thymi of both neonatal and adult offspring rats were aseptically acquired to scrutinize the effects of SEB on the Hh signaling pathway. It firstly found that prenatal SEB exposure clearly caused the increased expression of Shh and Dhh ligands of the Hh signaling pathway in thymus tissue of both neonatal and adult offspring rats, but significantly decreased the expression levels of membrane receptors of Ptch1 and Smo, transcription factor Gli1, as well as target genes of CyclinD1, C-myc, and N-myc in Hh signaling pathway of thymic T cells. These data suggest that prenatal SEB exposure inhibits the Hh signaling pathway in thymic T lymphocytes of the neonatal offspring, and this effect can be maintained in adult offspring via the imprinting effect.

Combined targeting of Hedgehog/GLI1 and Wnt/ß-catenin pathways in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a rare and aggressive form of non-Hodgkin lymphoma. Challenges in its treatment include relapse, drug resistance, and a short survival period. The Hedgehog/GLI1 (Hh/GLI1) and Wnt/ß-catenin pathways are crucial in cancer cell proliferation, survival, and drug resistance, making them significant targets for anticancer research. This study aimed to assess the effectiveness of combining inhibitors for both pathways against MCL and investigate the underlying molecular mechanisms. The co-expression of key proteins from the Hh/GLI1 and Wnt/ß-catenin pathways was observed in MCL. Targeting the Hh/GLI1 pathway with the GLI1 inhibitor GANT61 and the Wnt/ß-catenin pathway with the CBP/ß-catenin transcription inhibitor ICG-001, dual-target therapy was demonstrated to synergistically suppressed the activity of MCL cells. This approach promoted MCL cell apoptosis, induced G0/G1 phase blockade, decreased the percentage of S-phase cells, and enhanced the sensitivity of MCL cells to the drugs adriamycin and ibrutinib. Both GANT61 and ICG-001 downregulated GLI1 and ß-catenin while upregulating GSK-3ß expression. The interaction between Hh/GLI1 and Wnt/ß-catenin pathways was mediated by GANT61-dependent Hh/GLI1 inhibition. Moreover, GLI1 knockdown combined with ICG-001 synergistically induced apoptosis and increased drug sensitivity of MCL cells to doxorubicin and ibrutinib. GANT61 attenuated the overexpression of ß-catenin and decreased the inhibition of GSK-3ß in MCL cells. Overall, the combined targeting of both the Hh/GLI1 and Wnt/ß-catenin pathways was more effective in suppressing proliferation, inducing G0/G1 cycle retardation, promoting apoptosis, and increasing drug sensitivity of MCL cells than mono treatments. These findings emphasize the potential of combinatorial therapy for treating MCL patients.

GLI1 polymorphisms influence remission rate and prognosis of young de novo acute myeloid leukemia patients treated with cytarabine-based chemotherapy.

Acute myeloid leukemia (AML) is a highly heterogeneous hematological malignancy. Cytarabine (Ara-C)-based chemotherapy is the primary treatment for AML, but currently known prognostic risk stratification factors cannot fully explain the individual differences in outcome of patients. In this article, we reported that patients with homozygous GLI1 rs2228224 mutation (AA genotype) had a significantly lower complete remission rate than those with GG wild type (54.17% vs.76.02%, OR = 1.993, 95% CI: 1.062-3.504, P = 0.031). GLI1 rs2229300 T allele carriers had remarkably shorter overall survival (513 vs. 645 days, P = 0.004) and disease-free survival (342 vs. 456 days, P = 0.033) than rs2229300 GG carriers. Rs2229300 G > T variation increased the transcriptional activity of GLI1. CCND1, CD44 and PROM1 were potential target genes differentially regulated by GLI1 rs2229300. Our results demonstrated for the first time that GLI1 polymorphisms influence chemosensitivity and prognosis of young de novo AML patients treated with Ara-C.

Effects of mesenchymal stem cells versus curcumin on sonic hedgehog signaling in experimental model of Hepatocellular Carcinoma.

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.

GLI1 interaction with p300 modulates SDF1 expression in cancer-associated fibroblasts to promote pancreatic cancer cells migration.

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.

SUFU promotes GLI activity in a Hedgehog-independent manner in pancreatic cancer.

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.

Laser-assisted topical delivery of vismodegib reduces hedgehog gene expression in human basal cell carcinomas in vivo.

BACKGROUND: Systemically delivered hedgehog inhibitors including vismodegib and sonidegib are widely used to treat basal cell carcinomas (BCCs). Ablative fractional laser (AFL)-assisted topical delivery of vismodegib has been demonstrated in preclinical studies. The aim of this explorative clinical study was to evaluate intratumoral vismodegib concentrations and effect on hedgehog pathway gene expression following AFL-assisted topical vismodegib delivery to BCCs. METHODS: In an open-label clinical trial, 16 nodular BCCs (in n = 9 patients) received one application of CO2 -AFL (40 mJ/microbeam, 10% density) followed by topical vismodegib emulsion. After 3-4 days, vismodegib concentrations in tumor biopsies (n = 15) and plasma were analyzed and compared with samples from patients receiving oral treatment (n = 3). GLI1, GLI2, PTCH1, and PTCH2 expression was determined by quantitative polymerase chain reaction (n = 7) and GLI1 additionally by in situ hybridization (n = 3). RESULTS: Following AFL-assisted topical administration, vismodegib was detected in 14/15 BCCs and reached a median concentration of 6.2 µmol/L, which compared to concentrations in BCC tissue from patients receiving oral vismodegib (9.5 µmol/L, n = 3, p = 0.8588). Topical vismodegib reduced intratumoral GLI1 expression by 51%, GLI2 by 55%, PTCH1 and PTCH2 each by 73% (p ≤ 0.0304) regardless of vismodegib concentrations (p ≥ 0.3164). In situ hybridization demonstrated that GLI1 expression was restricted to tumor tissue and downregulated in response to vismodegib exposure. CONCLUSION: A single AFL-assisted topical application of vismodegib resulted in clinically relevant intratumoral drug concentrations and significant reductions in hedgehog pathway gene expressions.