Ablative fractional laser treatment reduces hedgehog pathway gene expression in murine basal cell carcinomas.

This study aimed to investigate the impact of ablative fractional laser (AFL) on hedgehog pathway gene expression in murine microscopic basal cell carcinomas (BCCs) and compare these results to the effect of topical treatment with vismodegib, an FDA-approved hedgehog inhibitor. In 25 mice, 1 cm2 skin test sites (n = 44) containing microscopic BCCs were exposed to one of three interventions: a single CO2 AFL treatment (1 pulse, 40 mJ/microbeam, wavelength 10.6 µm, 5% density, pulse rate 250 Hz, n = 12), eight topical vismodegib treatments (3.8 mg/mL, n = 8), or combination of AFL and vismodegib treatments (n = 9). Untreated controls were included for comparison (n = 15). After 4 days, skin samples were analyzed for hedgehog gene expression (Gli1, Gli2, and Ptch1) by qPCR and vismodegib concentrations by liquid chromatography mass spectrometry (data analyzed with two-tailed t-tests and linear regression). A single treatment with AFL monotherapy significantly reduced hedgehog gene expression compared to untreated controls (Gli1 72.4% reduction, p = 0.003; Gli2 55.2%, p = 0.010; Ptch1 70.9%, p < 0.001). Vismodegib treatment also reduced hedgehog gene expression (Gli1 91.6%; Gli2 83.3%; Ptch1 83.0%), significantly surpassing AFL monotherapy for two out of three genes (Gli1, p = 0.017; Gli2, p = 0.007; Ptch1, p = 0.15). AFL and vismodegib combination mirrored the effects of vismodegib monotherapy (Gli1, p = 0.424; Gli2, p = 0.289; Ptch1, p = 0.593), possibly due to comparable cutaneous vismodegib concentrations (mean ± SD, vismodegib monotherapy 850 ± 475 µmol/L; combination 1036 ± 824 µmol/L; p = 0.573). In conclusion, a single AFL treatment significantly reduced hedgehog gene expression in murine BCCs mimicking the effects of eight topical applications of vismodegib. Further studies are needed to assess whether AFL can be utilized for BCC treatment, either as monotherapy or in combination with other drugs.

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.

FUNDC2, a mitochondrial outer membrane protein, mediates triple-negative breast cancer progression via the AKT/GSK3ß/GLI1 pathway.

Triple-negative breast cancer (TNBC) lacks effective therapeutic targets and has a poor prognosis, easy recurrence and metastasis. It is urgent and important to explore TNBC treatment targets. Through mass spectrometry combined with qRT-PCR validation in luminal A cells and TNBC cells, high-content screening and clinical sample analysis, FUNDC2 was discovered as a novel target. The function of the outer mitochondrial membrane protein FUNDC2 in breast cancer is still unclear. In this study, we find that FUNDC2 expression in TNBC tissues is significantly higher than that in luminal subtype breast cancer tissues. FUNDC2 silencing in TNBC cells significantly reduces cell proliferation, migration and invasion. As demonstrated in vivo using subcutaneous tumor xenografts in mice, FUNDC2 suppression significantly inhibits tumor growth. The underlying mechanism might be mediated by inactivating its downstream signal AKT/GSK3ß and GLI1, a key factor of the Hedgehog signaling pathway. Therefore, FUNDC2 may promote TNBC progression and provide a therapeutic target for treating TNBC.

The Critical Role of The Piezo1/ß-catenin/ATF4 Axis on The Stemness of Gli1+ BMSCs During Simulated Microgravity-Induced Bone Loss.

Disuse osteoporosis is characterized by decreased bone mass caused by abnormal mechanical stimulation of bone. Piezo1 is a major mechanosensitive ion channel in bone homeostasis. However, whether intervening in the action of Piezo1 can rescue disuse osteoporosis remains unresolved. In this study, a commonly-used hindlimb-unloading model is employed to simulate microgravity. By single-cell RNA sequencing, bone marrow-derived mesenchymal stem cells (BMSCs) are the most downregulated cell cluster, and coincidentally, Piezo1 expression is mostly enriched in those cells, and is substantially downregulated by unloading. Importantly, activation of Piezo1 by systemically-introducing yoda1 mimics the effects of mechanical stimulation and thus ameliorates bone loss under simulated microgravity. Mechanistically, Piezo1 activation promotes the proliferation and osteogenic differentiation of Gli1+ BMSCs by activating the ß-catenin and its target gene activating transcription factor 4 (ATF4). Inhibiting ß-catenin expression substantially attenuates the effect of yoda1 on bone loss, possibly due to inhibited proliferation and osteogenic differentiation capability of Gli1+ BMSCs mediated by ATF4. Lastly, Piezo1 activation also slightly alleviates the osteoporosis of OVX and aged mice. In conclusion, impaired function of Piezo1 in BMSCs leads to insufficient bone formation especially caused by abnormal mechanical stimuli, and is thus a potential therapeutic target for osteoporosis.

Vemurafenib activates the sonic hedgehog pathway and promotes thyroid cancer stem cell self-renewal.

B-Raf kinase inhibitors such as vemurafenib (PLX4032) and dabrafenib have limited therapeutic efficacy on BRAF-mutated thyroid cancer. Cancer stem cells (CSCs) play important roles in tumor recurrence, drug resistance, and metastasis. Whether CSCs play a role in dampening the antitumor activity of B-Raf kinase inhibitors remains unknown. Here, we report that vemurafenib (PLX4032) induced the expression of several stemness-related genes including Gli1, Snail, BMI1, and SOX2 in two anaplastic thyroid cancer cell lines, SW1736 and 8505C, but decreased the expression of these genes in A375 cells, a human melanoma cell line. PLX4032 promoted thyroid cancer stem cell self-renewal, as evidenced by increased numbers of aldehyde dehydrogenase-positive cells and thyrospheres. Mechanistically, PLX4032 activates the PI-3 and mitogen-activated protein kinase pathways through HER3 to cross-activate Gli1, a transcription factor of the sonic hedgehog (Shh) pathway. GANT61, a specific inhibitor of Gli1, blocked the expression of the stemness-related genes in PLX4032-treated thyroid cancer cells in vitro and in vivo in two thyroid cancer xenograft models. GANT61 treatment alone weakly inhibited SW1736 tumor growth but enhanced the antitumor activity of PLX4032 when used in combination. Our study provides mechanistic insights into how thyroid cancer poorly responds to B-Raf kinase inhibitors and suggests that targeting B-Raf and the Shh pathway in combination may overcome thyroid cancer drug resistance.

Molecular targeting of the UDP-glucuronosyltransferase enzymes in high-eukaryotic translation initiation factor 4E refractory/relapsed acute myeloid leukemia patients: a randomized phase II trial of vismodegib, ribavirin with or without decitabine.

Drug resistance underpins poor outcomes in many malignancies including refractory and relapsed acute myeloid leukemia (R/R AML). Glucuronidation is a common mechanism of drug inactivation impacting many AML therapies, e.g., cytarabine, decitabine, azacytidine and venetoclax. In AML cells, the capacity for glucuronidation arises from increased production of the UDP-glucuronosyltransferase 1A (UGT1A) enzymes. UGT1A elevation was first observed in AML patients who relapsed after response to ribavirin, a drug used to target the eukaryotic translation initiation factor eIF4E, and subsequently in patients who relapsed on cytarabine. UGT1A elevation resulted from increased expression of the sonic-hedgehog transcription factor GLI1. Vismodegib inhibited GLI1, decreased UGT1A levels, reduced glucuronidation of ribavirin and cytarabine, and re-sensitized cells to these drugs. Here, we examined if UGT1A protein levels, and thus glucuronidation activity, were targetable in humans and if this corresponded to clinical response. We conducted a phase II trial using vismodegib with ribavirin, with or without decitabine, in largely heavily pre-treated patients with high-eIF4E AML. Pre-therapy molecular assessment of patients' blasts indicated highly elevated UGT1A levels relative to healthy volunteers. Among patients with partial response, blast response or prolonged stable disease, vismodegib reduced UGT1A levels, which corresponded to effective targeting of eIF4E by ribavirin. In all, our studies are the first to demonstrate that UGT1A protein, and thus glucuronidation, are targetable in humans. These studies pave the way for the development of therapies that impair glucuronidation, one of the most common drug deactivation modalities. Clinicaltrials.gov: NCT02073838.

Differential role of Pax6 and its interaction with Shh-Gli1-IDH2 axis in regulation of glioma growth and chemoresistance.

Glioma is a major brain tumor, and the associated mortality rate is very high. Contemporary therapies provide a chance of survival for 9-12 months. Therefore, a novel approach is essential to improve the survival rate. Sonic hedgehog (Shh) cell signaling is critical for early development in various tumors. This investigation attempted to explore the potential interaction and regulation of Shh-Gli1 cell signaling in association with paired box 6 (Pax6) and isocitrate dehydrogenase 2 (IDH2). The expression pattern of Shh, Gli1, Pax6, and IDH2 was examined by transcriptome analysis, immunohistochemistry, and confocal images. The results suggest the interaction of Shh-Gli1 cell signaling pathway with Pax6 and IDH2 and potential regulation. Thereafter, we performed protein-protein docking and molecular dynamic simulations (MDS) of Gli1 with Pax6 and IDH2. The results suggest differential dynamic interactions of Gli1-IDH2 and Gli1-Pax6. Gli1 knockdown downregulated the expression of Pax6 and upregulated the expression of IDH2. Moreover, Gli1 knockdown decreased the expression of the drug resistance gene MRP1. The knockdown of Pax6 gene in glioma cells downregulated the expression of Gli1 and IDH2 and promoted cell proliferation. Moreover, the efficacy of the treatment of glioma cells with temozolomide (TMZ) and Gli1 inhibitor GANT61 was higher than that of TMZ alone. MDS results revealed that the interactions of Gli1 with IDH2 were stronger and more stable than those with Pax6. Intriguingly, inhibition of Pax6 promoted glioma growth even in the presence of TMZ. However, the tumor-suppressive nature of Pax6 was altered when Gli1 was inhibited by GANT61, and it showed potential oncogenic character, as observed in other cancers. Therefore, we conclude that Pax6 interacted with IDH2 and Gli1 in glioma. Moreover, the Shh-Gli1-IDH2/Pax6 cell signaling axis provides a new therapeutic approach for inhibiting the progression of the disease and mitigating drug resistance in glioma.

GSK-3ß suppression upregulates Gli1 to alleviate osteogenesis inhibition in titanium nanoparticle-induced osteolysis.

Wear particle-induced periprosthetic osteolysis (PPO) have become a major reason of joint arthroplasty failure and secondary surgery following joint arthroplasty and thus pose a severe threat to global public health. Therefore, determining how to effectively suppress particle-induced PPO has become an urgent problem. The pathological mechanism involved in the PPO signaling cascade is still unclear. Recently, the interaction between osteogenic inhibition and wear particles at the implant biological interface, which has received increasing attention, has been revealed as an important factor in pathological process. Additionally, Hedgehog (Hh)-Gli1 is a crucial signaling cascade which was regulated by multiple factors in numerous physiological and pathological process. It was revealed to exert a crucial part during embryonic bone development and metabolism. However, whether Hh-Gli1 is involved in wear particle-induced osteogenic inhibition in PPO remains unknown. Our present study explored the mechanism by which the Hh-Gli1 signaling cascade regulates titanium (Ti) nanoparticle-induced osteolysis. We found that Hh-Gli1 signaling was dramatically downregulated upon Ti particle treatment. Mechanistically, glycogen synthesis kinase 3ß (GSK-3ß) activation was significantly increased in Ti particle-induced osteogenic inhibition via changes in GSK-3ß phosphorylation level and was found to participate in the posttranslational modification and degradation of the key transcription factor Gli1, thus decreasing the accumulation of Gli1 and its translocation from the cytoplasm to the nucleus. Collectively, these findings suggest that the Hh-Gli1 signaling cascade utilizes a GSK3ß-mediated mechanism and may serve as a rational new therapeutic target against nanoparticle-induced PPO.

Delivery siRNA with a novel gene vector for glioma therapy by targeting Gli1.

BACKGROUND: Gene therapy has recently shown considerable clinical benefit in cancer therapy during the past few years, and the application of this choice in cancer treatments is increasing continually. Gli1 is an ideal candidate target for cancer gene therapy and is important for tumorigenesis. METHODS: In this study, we developed a novel gene delivery system with a self-assembly method by using a 1,2-dioleoyl-3-trimethylammonium-propane and methoxy poly (ethylene glycol)-poly(lactide) copolymer (DMP), with zeta potential of 32.7 mV and measuring 35.6 nm. The effect of this delivery system was tested in vitro and in vivo. RESULTS: DMP showed good performance in delivering siRNA to glioma cells in vitro with high transfection performance (98%). Moreover, DMP-Gli1si shows a satisfactory anti-glioma effect via induction of cell apoptosis and cell growth inhibition in vitro. Furthermore, for subcutaneous tumor-bearing mice, treatment with the DMP-Gli1si complex significantly inhibited tumor growth by inhibiting Gli1 protein expression, promoting apoptosis, and reducing proliferation. CONCLUSION: The complex of Gli1 siRNA and DMP may potentially play an important role as a new drug in the clinical treatment of gliomas.