Sirt1 sustains sonic hedgehog signaling to promote medulloblastoma progression through regulating Gli3 processing

Sirt1 sustains sonic hedgehog signaling to promote medulloblastoma progression through regulating Gli3 processing | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Sirt1 sustains sonic hedgehog signaling to promote medulloblastoma progression through regulating Gli3 processing Yuan Wang, Jian Hu, Chaonan Zheng, Yingying Feng, Yuhang Li, Panpan Pei, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7344210/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Apr, 2026 Read the published version in Oncogene → Version 1 posted 8 You are reading this latest preprint version Abstract Medulloblastoma (MB), the most common malignant pediatric brain tumor with poor prognosis, high recurrence, and severe treatment-related toxicities. One-third of MB are driven by aberrant activation of the Sonic hedgehog (SHH) signaling pathway. In current study, through analysis of clinical patient cohorts and animal model database, and utilizing genetically engineered primary and xenograft mouse MB models, we investigated the role of Sirtuin1 (Sirt1), a class III histone deacetylase (HDAC), in SHH signaling and MB. We found that Sirt1 was highly expressed in both human and mouse SHH-type MB, and its expression positively correlated with SHH pathway activity and tumor proliferation. Knockdown of Sirt1 in primary MB cells significantly suppressed SHH signaling and MB proliferation in vitro , further impaired neoplastic progression and extended survival in orthotopic transplantation MB model. Mechanistically, we discovered that Sirt1 modulates SHH signaling at downstream by interacting with and deacetylating full-length Gli3 (Gli3FL), thereby inhibiting its proteolytic processing into the repressor form (Gli3R), which attenuates the negative feedback regulation of SHH signaling, sustaining pathway activation and promoting tumor progression. Importantly, pharmacological inhibition of Sirt1 demonstrated promising therapeutic efficacy in both subcutaneous transplantation and primary MB models. Our findings identify Sirt1 as a potential therapeutic target for SHH-driven MB and other cancers. Biological sciences/Cancer/CNS cancer Biological sciences/Molecular biology/Post-translational modifications/Acetylation Biological sciences/Cell biology/Mechanisms of disease Medulloblastoma SHH signaling Sirt1 Gli3 processing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Medulloblastoma (MB) is the most common primary brain malignancy affecting predominantly on children under 14 years of age, and accounts for approximately one-third of all pediatric brain tumors ( 1 ). MB almost exclusively occurs in the cerebellum and is characterized by rapid tumor growth and a high propensity for metastasis with high mortality, poor prognosis, and a significant recurrence rate. Currently, the primary treatment strategy involves a combination of surgery, radiotherapy, and chemotherapy ( 2 ), however, this approach demonstrates limited efficacy with severe side effects ( 3 ). Based on distinct molecular and pathological features, MB was traditionally classified into four molecular subtypes: WNT, SHH, Group 3, and Group 4, which are updated into WNT, SHH /TP53 wild-type, SHH/TP53 mutant, and non-WNT/non-SHH type recently ( 4 , 5 ). Among these, SHH-type MB, which results from aberrant activation of the Sonic Hedgehog (SHH) signaling pathway, constitutes approximately 30% of all diagnosed cases and, together with Group 3-MB, represent clinically refractory subtypes ( 6 ). SHH signaling pathway plays a pivotal role in organismal development, cell proliferation, and tissue differentiation ( 7 , 8 ). Activation of the canonical SHH signaling pathway is triggered by the binding of SHH ligand to its antagonistic receptor Patched 1 (Ptch1), which leads to the release of the transmembrane protein Smoothened (Smo). Thus, Smo is activated and facilitates the nuclear translocation of the Gli (Glioma-associated homologue) transcription factors family from the cytoplasm, thereby initiates the transcription of SHH signaling pathway target genes, including Cyclin D1 , N-Myc , Ptch1 , Ptch2 , Sfrp1 , and Gli itself ( 9 , 10 ). The Gli family in mammals consists of three members: Gli1, Gli2, and Gli3. Gli1 and Gli2 primarily function as transcriptional activators that positively regulate SHH signaling. In contrast, Gli3 contains a proteolytic processing determinant domain (PDD), which leads to the ubiquitination and proteolytic cleavage of full-length Gli3 protein (Gli3FL) following post-translational modifications such as acetylation and phosphorylation, consequently resulting in the formation of its repressor form (Gli3R, the N-terminal truncated fragment of Gli3FL). Gli3R competes with Gli1/2 for binding to target gene promoters but lacks transcriptional activation capability, thereby suppressing downstream gene expression, functionally, acts as a negative feedback on SHH signaling ( 11 ). Gli3R inhibited the proliferation of SHH-type MB via such negative feedback mechanism ( 12 ). The SHH signaling pathway is also implicated in the pathogenesis of various cancers, including multiple myeloma, colon cancer, pancreatic cancer, basal cell carcinoma, and prostate cancer, as well as SHH-type MB ( 13 – 15 ). Therefore, targeting this pathway may represent an important anti-tumor therapy approach. Current available SHH pathway inhibitors predominantly target to Smo protein, which frequently acquires resistance-conferring mutations upon treatment, leading to drug resistance and high recurrence rates ( 16 , 17 ). This necessitates the development of novel therapeutic strategies targeting SHH signaling pathway to overcome the existing treatment limitations. SHH-type MB originates from cerebellar granule neuron precursors (GNPs) ( 18 ). During early cerebellar development, active SHH signaling drives GNP expansion. As cerebellar development progresses, SHH signaling in GNPs gradually diminishes, GNPs cease proliferation and subsequently differentiate into mature granule neurons (GNs) ( 19 , 20 ). Aberrant activation of SHH pathway leads to the sustained and excessive proliferation of GNPs that ultimately drives SHH-type MB tumorigenesis. This pathological activation typically attributes to genetic alterations in SHH pathway components. For instance, SHH-type MB patients have shown Ptch1 deletions or inactivating mutations (~ 43%), Smo activating mutations (9%), or copy number gains of Gli1/2 (9%) ( 1 ). The currently available Smo inhibitors are only able to treat MB with the upstream SHH pathway mutations, targeting downstream signaling molecules of the SHH pathway would offer a broader clinical applicability for SHH-type MB and other cancers mediated by this pathway. Sirt1, a member of the seven-protein Sirtuins family, is a class III HDAC. Given the discovery of non-histone protein deacetylation, HDACs have recently been reclassified as lysine deacetylases (KDACs) ( 21 ). Sirtuins regulate gene expression and participate in diverse cellular processes, including DNA repair, metabolism, oxidative stress response, mitochondrial function, and biogenesis. Dysregulation of their expression or function may contribute to various human diseases, such as cancer, neurodegenerative diseases, metabolic disorders, and cardiovascular diseases ( 22 ). Among the Sirtuins family, Sirt1 is the most extensively studied member and can deacetylate numerous histone and non-histone proteins ( 23 ). Studies have demonstrated that Sirt1 is highly expressed in multiple human cancer cell lines and tumor tissues, such as prostate cancer, acute myeloid leukemia, and primary colon cancer ( 24 – 26 ). It is interesting to note that expression of Sirt1 was reported to be elevated in human MB cell lines, and its knockdown induces cell cycle arrest in vitro ( 27 ). While, another study demonstrated that the BCL6/BCOR/Sirt1 complex suppresses MB by inhibiting SHH signaling and promoting neurogenesis ( 28 ). Moreover, emerging information have implicated the involvement of Sirt1 in SHH signaling regulation, for instance, Sirt1 was shown to mediate resveratrol-induced SHH pathway activation ( 29 ); It was also shown to contribute to SHH-dependent microglial activation ( 30 ); Sirt1 may enhance SHH pathway activation in multiple myeloma by deacetylating Gli2 ( 31 ). Notably, in our previous study, Sirtuins inhibitors showed anti-proliferation effects on MB cells in vitro ( 32 ). In this report, we explore the role of Sirt1 on regulating of SHH signaling and its underlined molecular events associated with cell proliferation and tumor progression of SHH-type MB. Materials and Methods Animals Wild-type (WT) C57BL/6 mice and CB17/SCID mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Math1-Cre/Ptch1 loxp/loxp mice were bred in our laboratory as described previously ( 33 ). In these mice, Ptch1 is conditionally knocked out in Math1-positive cells, which are MB original cells, GNPs. These mice can spontaneously develop MB starting from 4–6 weeks old. Mice with mixed genders were used in our study. All mice were housed in a specific pathogen-free facility at Soochow University. All animal experiments were approved by and conducted in accordance with the Ethical and Welfare Committee of Soochow University. Cell culture HEK293T and NIH3T3 cells were obtained from Cell Bank/Stem Cell Bank, Chinese Academy of Sciences. Mouse embryonic fibroblasts (MEFs), murine MB cell line and DaoY cells were kindly provided by Dr. Zeng-jie Yang. SmoA1/Gli1/Gli2 stably overexpressed MEF cell lines ( SmoA1/Gli1/Gli2 -MEF) were constructed by lentivirus transfection in our lab. DaoY cells were maintained in Minimum Essential Medium (MEM, BasalMedia Technologies, Shanghai, China). All other cells listed above were cultured in DMEM (HyClone, Logan, Utah, USA) containing 10% FBS (Gibco, Grand Island, New York, USA) and 1% penicillin/streptomycin (Beyotime, Shanghai, China). For primary GNP and MB cell isolation and culture, we followed a previously described protocol ( 33 , 34 ). Briefly, cerebella or MB tumor tissues were dissected from WT mice at postnatal day 7 (P7) or tumor-bearing Math1-Cre/Ptch1 loxp/loxp mice, respectively, tissues were digested in a buffer containing 250 U/mL DNase (Sigma, St. Louis, Missouri, USA), 10 U/mL papain (Worthington, Lakewood, New Jersey, USA) and 200 µg/mL L-cysteine (Sigma) for 30 minutes at 37°C to acquire a single-cell suspension. The suspension was then centrifuged through a 35–65% Percoll gradient (Sigma). Cells from the 35–65% interface were collected and cultured in NB-B27 (Neurobasal with B27 supplement, 2 mM L-glutamine, 1 mM sodium pyruvate, and 1% penicillin/streptomycin). Chemical and protein reagents Recombinant N terminal of SHH protein (SHH-N) was purchased from Novoprotein (Beijing, China). In certain experiments, NIH3T3 cells were treated with 3 µg/mL SHH-N for indicated hours. Sirt1 inhibitors, Tenovin-1 and EX-527 (both from Selleck, Houston, Texas, USA), were used at indicated doses for cell treatment in vitro . For treatment of tumor-bearing mice in vivo , Tenovin-1 (90 mg/kg) and EX-527 (80 mg/kg) was administered by intraperitoneal injections. Immunohistochemistry and Immunofluorescent staining For immunohistochemistry, normal cerebella or subcutaneous allografts were dissected and then fixed in 4% PFA for 24 hours at 4°C. The preparation and immunohistochemistry staining of paraffin sections were performed by Servicebio Technology (Wuhan, China). Primary antibodies used for immunohistochemistry include anti-Sirt1 (1:200, Santa Cruz, Dallas, Texas, USA) and anti-Ki67 (1:200, Abcam, Waltham, Massachusetts). For immunofluorescent staining, tissues were harvested and frozen in Tissue Tek-OCT (Thermo Fisher Scientific, Waltham, Massachusetts), and then sectioned at 10–12 µm thickness. Sections were blocked in blocking buffer, PBS containing 0.2% Triton X-100 (Biosharp, Hefei, China) and 1% bovine serum albumin (BSA, Fisher bioreagents, Waltham, Massachusetts, USA), for 1 hour at room temperature, and incubated with primary antibodies overnight at 4 ºC. Then, sections were exposed to fluorescein-conjugated secondary antibodies for 2 hours at room temperature on next day. DAPI (Beyotime) was used to counter-stain nuclei for 10 minutes. Cell immunofluorescent staining followed the same protocol. The primary antibodies include anti-Ki67 (1:500), anti-Sirt1 (1:200), anti-HA (1:1000, Proteintech, Wuhan, China) and anti-Tuj1 (1:500, Abcam). The secondary antibodies include Alexa Fluor 488-goat anti-rabbit IgG (1:500, Proteintech), Alexa Fluor 488-goat anti-mouse IgG (1:500, Proteintech), Alexa Fluor 594-goat anti-mouse IgG (1:500, Proteintech) and Alexa Fluor 594-goat anti-rabbit IgG (1:500, Proteintech). Western blotting and immunoprecipitation Western blotting and immunoprecipitation Tissue homogenate or cell lysate was prepared in RIPA buffer (Thermo Fisher Scientific) containing protease and phosphatase inhibitor cocktail (Beyotime), and equal amounts of protein samples were loaded and separated by SDS-PAGE and subsequently transferred onto PVDF membranes (Millipore, Burlington, Massachusetts, USA). The membranes were blocked with blocking buffer (TBS containing 0.05% Tween-20 and 5% defatted milk powder) and incubated overnight at 4°C with primary antibodies against Gli1 (1:1000, Cell Signaling Technology, CST, Danvers, Massachusetts, USA), SHH (1:1000, CST), GAPDH (1:1000, CST), Sirt1 (1:500), Gli3 (1:1000, R&D Systems, Minneapolis, Minnesota, USA), HA (1:3000) and acetylated-Lysine (1:1000, CST). Then the membranes were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibodies, anti-rabbit IgG (1:5000, CST) and anti-mouse IgG (1:5000, CST), at room temperature for 2 hours on the following day. Protein bands were visualized using ECL substrate (Beyotime) and exposed on ChemiScope 3300 mini (Clinx, Suzhou, China). For immunoprecipitation, cell lysate was incubated with anti-HA antibody (1:300) overnight at 4°C, and the mixture was incubated with Protein A/G beads (Genscript, Nanjing, China) for another 2 hours at 4°C on next day. The beads were washed with PBS for 3–5 times and resuspended in SDS loading buffer for Western blotting analysis. Real-time quantitative PCR (qPCR) Total RNA was isolated from tissue homogenate or cell lysate using TRIzol reagent (Vazyme Biotech, Nanjing, China) in RNase-free conditions. cDNA was synthesized using RNA reverse transcription kit (Vazyme Biotech). Then, qPCR reactions were performed using SYBR qPCR Master Mix (Vazyme Biotech). Genetic manipulation To knock down Sirt1 in MB and MEF cells, lentiviruses carrying shRNAs constructed in pLV[shRNA]-mCherry-U6 plasmid were produced following regular protocol in HEK293T cells, and the supernatant in the relevant medium was collected for cell infection. For gene overexpression in cells, murine SmoA1 ( Smo W539L , activate mutation of Smo); Gli1 ; Gli2 and Sirt1 were constructed into vector pLV-AcGFP; Gli3 (full length, WT or K773R; K779R; K784R and K773/779/784R mutants) and Gli3R were constructed into vector pLV-HA. Lentiviruses carrying indicated genes listed above were produced to infect primary MB, MEF, NIH3T3 and HEK293T cells. Cell counting kit-8 (CCK-8) assay CCK-8 assay was performed to detect cell viability. Cells were inoculated into 96-well plates at 3 ⅹ10 3 / well density and cultured with indicated treatment for 48 hours. Then cells were incubated with 10 µL of CCK-8 reagent (Beyotime) for 1 hour to detect optical density at 450 nm wave length. Orthotopic and subcutaneous transplantation MB models Four- to five-week-old CB17/SCID mice were used for both models. For orthotopic transplantation model, primary MB cells were infected with a lentivirus carrying Sirt1 ShRNA or scrambled control, then mCherry + cells were sorted by flow cytometry 24 hours later, and 5×10 5 cells suspended in 5 µl PBS per mouse were intracranially injected into mice cerebellar. For subcutaneous transplantation model, mice were injected with 2×10 6 primary MB cells per mouse in the hind flanks subcutaneously. Tumor volume was measured and calculated as 1/2× (W 2 ×L) (W, width; L, length). When the tumor volume reached 200 mm 3 , the mice were randomly grouped and received treatments. The sirt1 inhibitor, Tenovin-1 (90 mg/kg bodyweight), or vehicle control was injected intraperitoneally into tumor-bearing mice daily at designated sites until the tumor size in the control group reached 1 cm 3 . Database analysis Relevant MB patient data were acquired from R2 database ( https://hgserver1.amc.nl/cgi-bin/r2/main.cgi ). Single-cell RNA sequencing data of SmoM2 -driven SHH-type MB was obtained from an open database ( http://gershon-lab.med.unc.edu/single-cell/ ). Statistics Experimental data were analyzed using GraphPad Prism software 9.5. Unpaired Student’s t test was used to calculate the difference in experiments containing two groups when data exhibit Gaussian distribution. Otherwise, data was analyzed via a non-parametric Student’s t test. For experiments containing three or more groups, one-way or two-way ANOVA was used to determine the levels of difference. Data are expressed as the mean ± SD. * P < 0.05 was considered to be significant. Results Sirt1 expression is upregulated in SHH-type MB and positively correlates with SHH signaling and MB proliferation In our previous study, epigenetic compounds have been screened targeting MB ( 32 ), we further identified Sirtuins inhibitors significantly suppressed MB cell proliferation in vitro (Fig. 1 A). To further investigate the functional role of Sirtuins in SHH signaling and MB tumorigenesis, we first detected the expression patterns of Sirtuin family members in primary MB cells and GNPs, the internal granular layer of normal adult mouse cerebellum served as the mature GNs control. Quantitative mRNA analysis revealed that Sirt1 was significantly highly expressed in MB cells and GNPs compared to GNs with the highest expression in MB cells (MB > GNPs > GNs); Sirt5 displayed a similar pattern but with much weaker expression; Remained other subfamilies of Sirtuins showed no significant changes across these cell types (Fig. 1 B). Meanwhile, detection of the protein expression by either immunohistochemistry or Western blotting confirmed the significant elevated expression of Sirt1 in tumor region compared to adjacent normal cerebellum (Fig. 1 C), so was in tumor cells versus GNPs/GNs (Fig. 1 D-E). It is known that MB cells exhibit hyperproliferative characteristics, GNPs maintain active proliferation, while GNs represent the terminally differentiated cells. We found that the activation status of the SHH signaling (as indicated by Gli1 protein levels) followed a consistent gradient: MB > GNPs > GNs, which were, intriguingly, in according with Sirt1 expressing pattern among these cell types, suggesting a potential role of Sirt1 in regulation of proliferation and SHH pathway activity (Fig. 1 D). Since it has been demonstrated that cultured MB cells exhibited progressive SHH pathway inactivation due to the lack of tumor microenvironment ( 32 , 35 ), we further showed the time-dependent SHH pathway inactivation in the cultured MB cells as evidenced by significant downregulation of SHH target genes ( Gli1 , Gli2 , Ptch2 , and Sfrp1 ), and the concomitant decreased Sirt1 expression (Fig. 1 F), which was in parallelled with the gradually ceased proliferation (Fig. 1 G-H), revealing a positive correlation between Sirt1 expression and cellular proliferation/SHH signaling activity. To exclude the possibility that this correlation may be specific to Ptch1 -deficient tumors, we analyzed single-cell RNA sequencing data from SmoM2 (active mutant of Smo )-driven MB mouse model. We observed that Sirt1 expressing population was predominantly localized to SHH-activated (Gli1+) cell clusters (Fig. 1 I). To further validate whether this Sirt1 expression pattern is clinically relevant, we analyzed Sirt1 mRNA levels in tumor tissues from patients across different MB subgroups via R2 database. Notably, Sirt1 expression was significantly elevated in SHH-type MB compared to other molecular subtypes (Fig. 1 J). These findings demonstrated that Sirt1 is preferentially overexpressed in SHH-driven MB, and its expression positively correlates with both proliferative status and SHH pathway activity. Sirt1 knockdown suppresses SHH signaling and MB cell proliferation To verify whether Sirt1 is involved in the regulation of MB proliferation, we first knocked down Sirt1 in primary MB cells and detect the tumor proliferation marker Ki67 by immunofluorescence staining. Compared with the scrambled control, the percentage of Ki67 + cells was significantly reduced in Sirt1 -knocked-down MB cells (Fig. 2 A-B). Meanwhile, Sirt1 knockdown markedly suppressed the expression of SHH pathway target genes (Fig. 2 C) and Gli1 protein expression (Fig. 2 D), indicating the inhibition of the SHH signaling. We next injected Sirt1 -knocked-down MB cells into the cerebellum of CB17/SCID mice to evaluate the effect of Sirt1 defect in an orthotopic MB xenograft model. As shown in Fig. 2 E-F, Sirt1 knockdown significantly inhibited MB formation and prolonged the survival of the experimental mice, confirming the importance of Sirt1 in SHH signaling and MB proliferation. NIH3T3 cells possess all key components of the SHH signaling pathway and can be activated by exogenous SHH ligands, making them a widely used model for studying SHH pathway regulation and mechanisms, we thus employed this cell line to investigate whether Sirt1 directly activates SHH signal transduction. NIH3T3 cells were stably infected with lentiviruses encoding either Sirt1 shRNA or Sirt1 cDNA. The transfecting efficacy was confirmed by Western blotting (Fig. 2 G). After treatment with recombinant SHH-N protein (N terminal of SHH ligand possessing bioactivity, 3 µg/mL), increased Gli1 levels was detected in WT NIH3T3 cells, confirming SHH pathway activation. While, Sirt1 -knockdown cells showed reduced Gli1 expression, revealing SHH-N stimulated SHH pathway activation was associated with Sirt1 expression. In support, Sirt1 -overexpressing cells exhibited the elevated Gli1 levels, which was further enhanced upon SHH-N treatment (Fig. 2 H-I). Sirt1 regulates SHH signaling downstream of Gli1/Gli2 by modulating Gli3 processing Activation of canonical SHH signaling pathway involves a cascade of sequential key signaling molecules: SHH ligand‒Ptch‒Smo‒Gli1/2. We already identified that Sirt1 functions downstream of SHH ligand and Ptch1 since NIH3T3 cells were under SHH ligand stimulation and primary MB cells carried Ptch1 gene deletion (Fig. 2 ). To further define Sirt1's action in the signaling cascade, we established three distinct SHH pathway-activated MEF cell lines by using a lentivirus-mediated stable overexpression system: SmoA1 -MEF (active Smo mutant, W539L ), Gli1 -MEF, and Gli2 -MEF. The efficiency of overexpression was confirmed by qPCR, and each of these genetic modifications effectively activated the SHH pathway independently (Supplementary Fig. 1). As shown in Fig. 3 A-D, Sirt1 knockdown in all three mutants as well as in WT MEF cells consistently downregulated SHH pathway target genes as compared to scrambled control. These results indicate that Sirt1 regulates SHH signaling cascades downstream of Gli1/2. Gli3FL undergoes processing to generate Gli3R, which negatively regulates the SHH pathway through competing with Gli1/Gli2. To investigate whether Sirt1 regulates this pathway by modulating Gli3 processing, we examined the protein levels of Gli3R and Gli3FL, as well as their ratio (Gli3R/Gli3FL) in NIH3T3 cells with either Sirt1 knockdown or overexpression. This ratio is considered to be an indicator of the extent of Gli3 processing. As shown in Fig. 3 E-F, in the absence of SHH ligand stimulation, no significant differences in Gli3 processing were observed among the experimental groups; In contrast, in the presence of SHH ligand stimulation, Sirt1 -knockdown cells exhibited a significant increase in the Gli3R/Gli3FL ratio, and as expected, Sirt1 -overexpressing cells decreased the ratio, clearly demonstrating that Sirt1 suppressed the proteolytic processing of Gli3. Similarly, Sirt1 -knockdown primary MB cells as well as in the orthotopic xenograft showed significantly elevated Gli3R/Gli3FL ratios (Fig. 3 G-H). To confirm whether Sirt1 maintains SHH pathway activity by inhibiting Gli3 processing, we performed rescue experiments in MEF cells. The results showed that Sirt1 overexpression alone was sufficient to activate SHH signaling, while co-expression of Gli3R abolished the Sirt-mediated SHH pathway activation (Fig. 3 I). Sirt1 directly interacts with and deacetylates Gli3 It was reported that Sirt1 directly interacts with Gli2 and mediates its deacetylation ( 31 ). Given the high homology between Gli2 and Gli3, we hypothesized that Sirt1 might similarly interact with and deacetylate Gli3. Due to the lack of reliable antibodies to immunoprecipitate endogenous Gli3 or Sirt1, we employed NIH3T3 cells transfected with either HA-tagged Gli3FL or HA-Gli3R. Co-IP experiments using anti-HA antibody demonstrated that endogenous Sirt1 specifically co-precipitated with Gli3FL, but not with Gli3R (Fig. 4 A). The observation was confirmed by immunofluorescence staining which revealed the colocalization between endogenous Sirt1 and exogenous HA-Gli3FL (Fig. 4 B). In cellular fractionation separations, Sirt1 was present in both nuclear and cytoplasmic compartments (Fig. 4 C). Given that Gli3FL predominantly localizes to the cytoplasm that is translocated in to the nucleus when processed to Gli3R, the observed cytoplasmic colocalization of Sirt1 with Gli3FL is in agreement with co-IP results. Given Sirt1 might itself be a direct target of SHH pathway ( 31 ), to eliminate potential confounding effects of SHH signaling on Sirt1 protein levels, we utilized HEK293T cells, which exhibit minimal basal SHH pathway activity, to establish a HA-Gli3FL stably expressing line. Sirt1 overexpression significantly suppressed Gli3 processing; while treatment with Sirt1 inhibitor Tenovin-1 (100 nM) promoted it (Fig. 4 D-E). We next assessed Gli3 acetylation status by co-IP, which revealed that Gli3 underwent endogenous acetylation (Fig. 4 F-G), and Tenovin-1 treatment increased Gli3 acetylation (Fig. 4 G), suggesting that Sirt1 indeed suppressed Gli3 acetylation. To further investigate the molecular mechanism by which Sirt1 regulates Gli3 processing, we analyzed the lysine acetyltransferase (KAT)-specific acetylation site database ( 36 ) and the homologous acetylation sites of Gli2. We predicted three potential acetylation sites on the Gli3 protein: K773, K770, and K784 (Fig. 4 H). Plasmids for each or all three of those lysine mutants (replaced by arginine) were prepared and transfected to HEK293T cells. As shown in Fig. 4 I-J, all single-site mutations reduced the Gli3R/Gli3FL ratio, while simultaneous mutation of all three sites led to a more dramatic decrease, indicating that these acetylation sites are critical for Gli3FL processing. To determine whether Sirt1 regulates Gli3 acetylation through these three sites, we overexpressed Sirt1 or applied its inhibitor in WT HA-Gli3FL - and mutant HA-Gli3FL (K773/779/784R) -cells, respectively. The results showed that Sirt1 overexpression suppressed Gli3 acetylation, while Sirt1 inhibitor treatment increased Gli3 acetylation in WT Gli3 overexpressing cells. In contrast, mutant Gli3 did not alter acetylation by Sirt1 overexpression or Sirt1 inhibition. We also observed that the acetylation levels of the K773/779/784R-mutated Gli3 were lower than those of the WT (Fig. 4 K-N). Taken altogether, our data indicate that Sirt1 deacetylates Gli3 at K773, K779, and K784 residues in the C-terminal transcriptional activation domain. Sirt1 inhibitors suppress MB cell proliferation in vitro So far, we have revealed that Sirt1 regulates the SHH signaling pathway by interacting with Gli3. We wondered whether selective pharmacological inhibitors of Sirt1 could suppress the proliferation of SHH-type MB. We selected two well-characterized Sirt1 inhibitors, Tenovin-1 and EX-527, and treated Math1-cre/Ptch1 loxp/loxp mice derived primary MB cells, cell viability exhibited that both inhibitors suppressed MB cell proliferation in a dose-dependent manner, with IC 50 values of 241.3 nM for Tenovin-1 and 1.2 µM for EX-527 (Fig. 5 A-B). Immunofluorescence staining revealed that Tenovin-1(1µM) or EX-527 (1µM) treatment significantly reduced the percentage of Ki67 + cells, while increasing the proportion of differentiated (Tuj1+) cells (Fig. 5 C-D). We further confirmed the anti-proliferative effects of these inhibitors in human SHH-MB cells (DAOY-1) and SmoA1 -overexpressing MB cell line. The SmoA1 mutation confers resistance to the Smo inhibitor Vismodegib ( 16 ). Consistent with the results in Ptch1 -deficient mouse MB cells, Tenovin-1 or EX-527 effectively suppressed proliferation in both DAOY-1 and Vismodegib-resistant SmoA1 -MB cells (Fig. 5 E-H). Pharmacological inhibition of Sirt1 attenuates MB progression in vivo We next assessed the therapeutic potential of Sirt1 inhibitors in vivo . Since Tenovin-1 has limited blood-brain barrier permeability, we employed a subcutaneous MB xenograft approach to evaluate its efficacy. Treatment with Tenovin-1 (90 mg/kg/day, administered via intraperitoneal injection) for 2 weeks markedly inhibited tumor growth (Fig. 6 A-B). Immunohistochemical analysis of tumor tissues confirmed that Tenovin-1 treatment significantly reduced Ki67 + cells (Fig. 6 C). As expected, Tenovin-1 treatment also suppressed SHH pathway target genes expression, but increased Gli3R/Gli3FL ratio in tumors (Fig. 6 D-E). We also tested EX-527, a highly specific Sirt1 inhibitor with demonstrated blood-brain barrier permeability ( 37 , 38 ), on primary MB in Math1-cre/Ptch1 loxp/loxp mice. We treated these mice with EX-527 (80 mg/kg/day via intraperitoneal injection), started from week 4 for 14 consecutive days. Immunofluorescence staining showed significantly reduced tumor cell proliferation (Ki67 + cells) in EX-527-treated mice compared to controls (Fig. 6 F-G). Western blotting analysis demonstrated attenuated Gli1 expression and enhanced Gli3 processing in primary tumors with EX-527 treatment (Fig. 6 H). Discussion Although significant progress has been made in elucidating the pathogenesis of MB, no significant clinical breakthroughs have been achieved ( 39 , 40 ). Therefore, there is an urgent need for more comprehensive investigations of MB pathogenesis mechanisms to development new therapeutic targets. In current study, we investigate the role and the underlying mechanism of Sirt1 in the SHH signaling pathway and SHH-type MB. We found that Sirt1 was highly expressed in both human and mouse SHH-type MB, and its expression positively correlated with SHH pathway activity and cell proliferation in MB cells. To investigate its role in MB, we knocked down Sirt1 in primary MB cells and observed a significant inhibition of MB proliferation in vitro and a reduction in SHH pathway activity. Moreover, MB cells with Sirt1 defect almost lost their tumor-forming ability when orthotopically transplanted into mouse cerebellum. These data indicate that Sirt1 positively regulates SHH signaling and MB proliferation both in vitro and in vivo . We further validated our findings using NIH3T3 cells, a tool cell line that possesses all components of the SHH pathway but requires exogenous SHH ligand for pathway activation. Knocking down Sirt1 in these cells also suppressed SHH pathway activation, confirming that Sirt1 directly regulates SHH pathway. To identify the specific cascade at which Sirt1 modulates the SHH signal pathway, we knocked down Sirt1 in MEF cells overexpressing SmoA1 , Gli1 , or Gli2 , respectively, and found that Sirt1 depletion significantly inhibited SHH pathway activation in all cases, suggesting that Sirt1 functions downstream of Gli1/2. Considering that Gli3 always acts as negative feedback of SHH signaling through processing into Gli3R ( 41 ) at the downstream of Gli1/2. Although Gli2 also contains a PDD, its processing into Gli2R is inefficient and typically does not contribute significantly to negative feedback regulation ( 42 ). Thus, in canonical SHH signaling, the relative abundance of Gli3R versus activated Gli2/Gli3 determines the net pathway activity ( 43 ). We then focused on investigating whether Sirt1 affects the generation of Gli3R. Results showed that Sirt1 knockdown in NIH3T3 cells significantly enhanced Gli3R production while reduced SHH pathway activity concurrently, whereas Sirt1 overexpression suppressed Gli3R generation and upregulated SHH signaling. To further confirm whether Sirt1 regulates the SHH pathway through modulating Gli3 processing, we overexpressed Sirt1 with or without co-overexpression of Gli3R in NIH3T3 cells, and found that overexpression of Sirt1 alone was sufficient to trigger SHH pathway activation, whereas co-expression of Gli3R dampened the activation, confirming that Sirt1 positively regulates SHH pathway by inhibiting Gli3 processing into Gli3R. Next, we explored the molecular mechanism by which Sirt1 inhibits Gli3 processing. Gli3 processing involves a multistep process, including acetylation, phosphorylation, and subsequent ubiquitin-proteasome-mediated degradation ( 44 – 46 ). This process is tightly regulated by diverse mechanisms ( 45 – 48 ), which may significantly influence both physiological and pathological SHH-mediated processes. For example: Cadherin-7 has been shown to enhance SHH signaling during neural tube development by suppressing Gli3 processing ( 49 ); Set7-mediated methylation of Gli3 promotes SHH pathway activation ( 50 ). Given the deacetylase property of Sirt1, and its action on deacetylating Gli2 ( 31 ), we wondered whether it may interact and deacetylate Gli3 directly. Co-IP experiments revealed that Sirt1 interacts with Gli3FL but not Gli3R, suggesting that Sirt1 may regulate the initial steps of Gli3 processing and that the interaction domain might be lost during Gli3R generation. To exclude the potential influence of SHH signaling on Sirt1 itself, we used SHH-inactive HEK293T cells to study the specific mechanism of Sirt1-mediated Gli3 processing regulation. Overexpression of Sirt1 as well as treatment with the Sirt1 enzymatic inhibitor, Tenovin-1, in these cells reproduced the previous observations: Sirt1 overexpression inhibited Gli3 processing, while the inhibitor promoted it. We then examined Sirt1’s effect on Gli3 acetylation in these cells. We found that overexpressed Gli3 in HEK293T cells underwent acetylation, which was enhanced by Sirt1 inhibition, indicating that Sirt1 suppresses Gli3 acetylation in a SHH pathway-independent manner. To verify whether Gli3 acetylation affects its processing, we mutated key lysine residues in Gli3 that are critical for acetylation, and overexpressed the mutants in HEK293T cells. The mutations drastically reduced Gli3 processing. Furthermore, in cells overexpressing the mutant Gli3, neither Sirt1 overexpression nor inhibitor treatment altered Gli3 acetylation levels, confirming that these residues are essential for Sirt1-mediated deacetylation of Gli3. Finally, we assessed the therapeutic feasibility of targeting Sirt1 in SHH-type MB. We evaluated two well-characterized Sirt1 inhibitors, Tenovin-1 and EX527. In vitro studies demonstrated that both inhibitors effectively suppressed proliferation across multiple MB cell models, including primary mouse MB cells, human SHH-type MB cells, and SmoA1 -overexpressing MB cells. Importantly, in vivo validation using distinct tumor models revealed consistent anti-tumor efficacy: Tenovin-1 treatment in subcutaneous xenografts significantly enhanced Gli3 processing, suppressed SHH pathway activation, and markedly inhibited tumor growth. Similarly, EX-527 administration in a genetically engineered primary MB model faithfully reproduced these anti-proliferative effects. The concordant therapeutic outcomes observed across both transplanted and spontaneous MB models strongly support Sirt1 as a novel and promising therapeutic target for SHH-type MB. The striking discovery of the present work revealed the critical role of Sirt1 in SHH signaling and SHH-type MB, and confirmed its action at the downstream of SHH pathway, making it a valuable and promising target for SHH-driven malignancies treatment. Since current clinical approaches predominantly employ Smo inhibitors, such as Vismodegib, as the principal pharmacological strategy. These agents are substantially limited by the rapid emergence of acquired resistance mediated by Smo mutations, frequently resulting in tumor relapse ( 16 , 17 ). Furthermore, Smo-targeted therapies are only effective against the subset of SHH-MB cases harboring upstream pathway alterations (approximately 50%). While targeting Sirt1 may overcome these limitations, which would potentially offer broader clinical applicability for both SHH-MB and other SHH-driven diseases. Our present work also elaborated that Sirt1 regulates SHH pathway by disrupting the negative feedback loop mediated by Gli3R. As established, genetic mutations serve as initiating events that activate oncogenic signaling pathways, sustained pathway activation is required to maintain tumor cell proliferation and facilitate the progression to full-grown tumors. In SHH-type MB, for instance, excessive SHH signaling triggered by genetic mutations such as Ptch1 deletion induces feedback inhibition through enhanced Gli3 processing into Gli3R. Sirt1 is demonstrated here acting as the “second hit” to breakdown this negative feedback and ultimately lead to the tumor formation and progression. Mechanistically, out work illustrated that Sirt1 suppresses Gli3 processing via post-translational modifications, which also emphasizes the emerging recognition of post-translational modifications and epigenetic regulation in SHH-type MB pathogenesis. Admittedly, the precise mechanisms underlying Sirt1-mediated regulation of the SHH pathway require further in-depth investigation. Several critical questions remain to be addressed: Whether the interaction between Sirt1 and Gli3 depends on specific acetylation sites or distinct binding domains; How the binding affinity between these proteins is regulated; And the exact binding regions and sites need to be systematically mapped using various truncated protein fragments. Furthermore, while Gli1 and Gli2 are predominantly nuclear proteins, Gli3FL mainly localizes to the cytoplasm. Interestingly, aberrant cytoplasmic accumulation of Sirt1 has been linked to enhanced protein stability regulated by PI3K/IGF-1R signaling ( 51 ). Does Sirt1 modulate SHH pathway activity by shuttling between the nucleus and cytoplasm? These aspects warrant thorough exploration to fully elucidate the crosstalk between Sirt1 and SHH signaling in MB pathogenesis. In summary, our findings support a mechanistic model wherein Sirt1-mediated deacetylation of Gli3 inhibits its proteolytic processing into the repressor form (Gli3R), consequently sustaining SHH pathway activation and promoting SHH-type MB proliferation. This study elucidates a previously unrecognized regulatory axis: Sirt1‒Gli3 processing‒SHH pathway activity, providing novel mechanistic insights into MB pathogenesis. Specifically, we demonstrate that Sirt1 potentiates SHH signaling through suppression of the Gli3R-mediated negative feedback loop, representing a critical post-translational regulatory layer in SHH-driven tumorigenesis. The present data also implicate Sirt1 as a potential target for SHH-type MB drug discovery. Declarations Ethics approval and consent to participate All animal experiments were carried out according to guidelines approved by the Institutional Animal Care and Use Committee of Soochow University. Competing interests The authors declare no competing interests. Authors' contributions JH: project management, research conduction, experiments performance, data analysis, results interpretation, and manuscript preparation. CNZ: research conduction, experiments performance, data analysis, and results interpretation. YYF/YHL/PPP/MY/YHQ: experiments performance, data acquisition. LZ/LTZ: data analysis, results interpretation, funding. ZXC: results interpretation, manuscript preparation, funding. YW: project management, research conduction, data analysis, results interpretation, manuscript preparation, funding. Acknowledgements This research was supported by National Natural Science Foundation of China (82373900 and 82073873 to Yuan Wang, 82072798 to Li Zhang, 81973334 to Xue-Chu Zhen, 32371016 and 31970909 to Long-Tai Zheng), the National Innovation of Science and Technology-2030 (Program of Brain Science and Brain-Inspired Intelligence Technology) (2021ZD0204004), the National Key Research and Development Program of China (2021YFE0206000) and the Priority Academic Program Development of the Jiangsu Higher Education Institutes (PAPD). Availability of data The data that support the findings of this study are available from corresponding author on reasonable request. References Northcott PA, Robinson GW, Kratz CP, Mabbott DJ, Pomeroy SL, Clifford SC, et al. Medulloblastoma. Nat Rev Dis Primers. 2019;5(1):11. Beccaria K, Padovani L, Bouchoucha Y, Doz F. Current treatments of medulloblastoma. Curr Opin Oncol. 2021;33(6):615-20. Northcott PA, Korshunov A, Pfister SM, Taylor MD. 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Supplementary Files Supplementarymaterials.docx Supplementary material Cite Share Download PDF Status: Published Journal Publication published 18 Apr, 2026 Read the published version in Oncogene → Version 1 posted Editorial decision: revise 30 Oct, 2025 Review # 1 received at journal 05 Sep, 2025 Reviewer # 2 agreed at journal 18 Aug, 2025 Reviewer # 1 agreed at journal 18 Aug, 2025 Reviewers invited by journal 12 Aug, 2025 Submission checks completed at journal 12 Aug, 2025 Editor assigned by journal 11 Aug, 2025 First submitted to journal 11 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7344210","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":499680300,"identity":"4b61a9fa-8e42-4007-b020-6bf39143b015","order_by":0,"name":"Yuan 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Zhen","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xue-Chu","middleName":"","lastName":"Zhen","suffix":""}],"badges":[],"createdAt":"2025-08-11 08:41:56","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7344210/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7344210/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41388-026-03781-1","type":"published","date":"2026-04-18T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89469403,"identity":"a2d2aec7-2f8e-466e-a858-45a789d2dcb3","added_by":"auto","created_at":"2025-08-20 09:15:05","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1270989,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSirt1 expression is upregulated in SHH-type MB and positively correlates with SHH signaling and MB proliferation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Epigenetic compounds for anti-MB proliferation drug screening (Numbers in parentheses indicate the quantity of effective compounds in each category). \u003cstrong\u003eB \u003c/strong\u003eExpression profiling of Sirtuin family (\u003cem\u003eSirt1\u003c/em\u003e-\u003cem\u003e7\u003c/em\u003e) genes in GNs (served as control), GNPs, and MB cells by qPCR analysis. \u003cstrong\u003eC\u003c/strong\u003e Representative immunohistochemistry staining of Sirt1 (in brown) in MB tumor-bearing cerebellum derived from \u003cem\u003eMath1-Cre/Ptch1\u003c/em\u003e\u003csup\u003e\u003cem\u003eloxp/loxp\u003c/em\u003e\u003c/sup\u003e mice. Normal adjacent tissue (N) and tumor region (T) was framed out and zoomed in. H\u0026amp;E: Hematoxylin-eosin staining. \u003cstrong\u003eD-E\u003c/strong\u003e Sirt1 and Gli1 protein levels was detected by Western blotting and the relative gray density of Sirt1 protein bands was quantified. \u003cstrong\u003eF-H\u003c/strong\u003e Primary MB cells were cultured for 0, 48 or 72 hours (h) \u003cem\u003ein vitro\u003c/em\u003e, the mRNA expression of SHH pathway target genes (\u003cem\u003eGli1\u003c/em\u003e, \u003cem\u003eGli2\u003c/em\u003e, \u003cem\u003ePtch2\u003c/em\u003e and \u003cem\u003eSfrp1\u003c/em\u003e) as well as \u003cem\u003eSirt1\u003c/em\u003e was evaluated by qPCR (\u003cstrong\u003eF\u003c/strong\u003e). Cell proliferative marker Ki67 was immuno-stained in red (\u003cstrong\u003eG\u003c/strong\u003e), DAPI counterstained nuclei, and the percentage of Ki67+ cells in DAPI+ cells was quantified (\u003cstrong\u003eH\u003c/strong\u003e). \u003cstrong\u003eI\u003c/strong\u003e Analysis of \u003cem\u003eSirt1\u003c/em\u003e and \u003cem\u003eGli1\u003c/em\u003e expression in single-cell RNA sequencing database of \u003cem\u003eSmoM2\u003c/em\u003e-driven SHH-type MB. \u003cstrong\u003eJ\u003c/strong\u003e Analysis of \u003cem\u003eSirt1 \u003c/em\u003eexpression of four subtype human MB in R2 database. ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 vs. GNs in panel \u003cstrong\u003eB\u003c/strong\u003e; ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 vs. 0 h in panel \u003cstrong\u003eF\u003c/strong\u003e and \u003cstrong\u003eH\u003c/strong\u003e; *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7344210/v1/522c8ff8e13b2523111b0da8.jpg"},{"id":89469407,"identity":"168fb3b8-8677-4b56-806a-2076fe6033b6","added_by":"auto","created_at":"2025-08-20 09:15:05","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":926889,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSirt1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e knockdown suppresses SHH signaling and MB cell proliferation \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand tumorigenesis \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrimary MB cells were infected with lentivirus carrying \u003cem\u003eSirt1\u003c/em\u003e shRNA or scrambled control for 48 hours. \u003cstrong\u003eA\u003c/strong\u003e Ki67 was immuno-stained in green, mCherry showed in red; \u003cstrong\u003eB\u003c/strong\u003e The percentage of Ki67+ in mCherry+ cells was quantified; \u003cstrong\u003eC\u003c/strong\u003e The mRNA expression of SHH signaling target genes (\u003cem\u003eGli1\u003c/em\u003e, \u003cem\u003eGli2\u003c/em\u003e, \u003cem\u003ePtch2\u003c/em\u003e and \u003cem\u003eSfrp1\u003c/em\u003e) and \u003cem\u003eSirt1\u003c/em\u003e were evaluated by qPCR; \u003cstrong\u003eD\u003c/strong\u003e Protein levels of Sirt1 and Gli1 were examined by Western blotting. \u003cstrong\u003eE-F\u003c/strong\u003e 5×10\u003csup\u003e5\u003c/sup\u003e primary MB cells of \u003cem\u003eSirt1\u003c/em\u003e knocking down (right) or control (left) were injected into the cerebella of \u003cem\u003eCB17/SCID\u003c/em\u003e mice. Five weeks post injection, tumor-bearing cerebella were dissected for sectioning stained with DAPI (\u003cstrong\u003eE\u003c/strong\u003e); the survival curve was plotted (\u003cstrong\u003eF\u003c/strong\u003e), n=6. \u003cstrong\u003eG-I\u003c/strong\u003e \u003cem\u003eSirt1\u003c/em\u003e was either knocked down or overexpressed (O/E) by lentivirus infection in NIH3T3 cells, the protein levels of Sirt1 were assessed by Western blotting (\u003cstrong\u003eG\u003c/strong\u003e). These cells were treated with recombinant SHH-N (3 µg/mL) or vehicle for 48 hours, Gli1 protein levels were evaluated by Western blotting (\u003cstrong\u003eH\u003c/strong\u003e) and the relative gray density was quantified (\u003cstrong\u003eI\u003c/strong\u003e).\u0026nbsp; ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 vs. Scrambled in panel \u003cstrong\u003eB\u003c/strong\u003e. ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7344210/v1/3873cd676a6468ef4685879f.jpg"},{"id":89470053,"identity":"08bcaf32-a5c1-476d-a237-d500c1fd2527","added_by":"auto","created_at":"2025-08-20 09:23:06","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":870996,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSirt1 regulates SHH signaling downstream of Gli1/Gli2 by modulating Gli3 processing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-D\u003c/strong\u003e \u003cem\u003eSirt1\u003c/em\u003ewas knocked down in \u003cem\u003eSmoA1\u003c/em\u003e, \u003cem\u003eGli1\u003c/em\u003e, or \u003cem\u003eGli2\u003c/em\u003e-overexpressing and WT MEF cells by shRNA, target genes of SHH signaling and \u003cem\u003eSirt1\u003c/em\u003e was evaluated by qPCR. \u003cstrong\u003eE\u003c/strong\u003e \u003cem\u003eSirt1\u003c/em\u003e was either knocked down or overexpressed (O/E) in NIH3T3 cells, cells were treated with recombinant SHH-N (3 µg/mL) or vehicle for 48 hours, the protein levels of Gli3FL and Gli3R were assessed by Western blotting. \u003cstrong\u003eF\u003c/strong\u003e The protein bands density ratio of Gli3R/Gli3FL in panel \u003cstrong\u003eE\u003c/strong\u003e was quantified. \u003cstrong\u003eG-H \u003c/strong\u003e\u003cem\u003eSirt1\u003c/em\u003ewas knocked down by shRNA in primary MB cells (\u003cstrong\u003eG\u003c/strong\u003e), and these cells were used to establish orthotopic xenograft tumor model, cerebella were dissected 5 weeks post MB cell inoculation (\u003cstrong\u003eH\u003c/strong\u003e), the protein levels of Gli3FL and Gli3R in MB cells (\u003cstrong\u003eG\u003c/strong\u003e) or xenograft tumor tissues (\u003cstrong\u003eH\u003c/strong\u003e) were assessed by Western blotting. \u003cstrong\u003eI\u003c/strong\u003e MEF cells were overexpressed with \u003cem\u003eSirt1\u003c/em\u003e alone or together with \u003cem\u003eGli3R\u003c/em\u003e, the expression of SHH signaling target genes were evaluated by qPCR. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ns: not significant.\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7344210/v1/1c9d36e2abb48817ca64c8fc.jpg"},{"id":89469408,"identity":"30e082e2-48da-4014-ad33-029213effabb","added_by":"auto","created_at":"2025-08-20 09:15:05","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":879529,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSirt1 directly interacts with and deacetylates Gli3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003eNIH3T3 cells overexpressed \u003cem\u003eHA-Gli3FL\u003c/em\u003e or \u003cem\u003eHA-Gli3R\u003c/em\u003e or vector control (HA) were treated with recombinant SHH-N (3 µg/mL) for48 hours. Cell lysate was prepared and immunoprecipitated with anti-HA antibody, then the blotting was performed using antibodies against the indicated targets. \u003cstrong\u003eB\u003c/strong\u003e Representative immunostaining of exogenous HA-Gli3FL (in green) and endogenous Sirt1 (in red) in NIH3T3 cells. \u003cstrong\u003eC\u003c/strong\u003eSubcellular fractionation and Western blotting analysis of Sirt1 distribution in cytoplasmic and nuclear fractions of NIH3T3 cells. \u003cstrong\u003eD-E\u003c/strong\u003e \u003cem\u003eHA-Gli3FL\u003c/em\u003estably overexpressing HEK293T cells were established and co-overexpressed with \u003cem\u003eSirt1\u003c/em\u003eor \u003cem\u003eGFP\u003c/em\u003e as negative control, or treated with Sirt1 inhibitor (Tenovin-1, 100 nM) for 48 hours. Western blotting analysis of Gli3FL and Gli3R protein (\u003cstrong\u003eD\u003c/strong\u003e) and quantification of Gli3R/Gli3FL ratio (\u003cstrong\u003eE\u003c/strong\u003e) were performed. \u003cstrong\u003eF-H\u003c/strong\u003e HEK293T cells were overexpressed with \u003cem\u003eHA-Gli3FL\u003c/em\u003e for 48 hours. The acetylation status of Gli3 was detected by anti-HA (upper panel) and anti-acetylated lysine (lower panel) after immunoprecipitation with antibodies against acetylated lysine (AcK, upper panel), HA (lower panel), or IgG control (\u003cstrong\u003eF\u003c/strong\u003e). Cells were treated with DMSO or Tenovin-1 (100 nM) for 48 hours, and the acetylation status of Gli3 was detected by blotting with anti-acetylated lysine after HA immunoprecipitation (\u003cstrong\u003eG\u003c/strong\u003e). Biochemical domains of Gli3 and prediction of its post-translational acetylation sites (\u003cstrong\u003eH\u003c/strong\u003e). \u003cstrong\u003eI-N\u003c/strong\u003e \u003cem\u003eGli3-WT\u003c/em\u003e and different mutant constructs (\u003cem\u003eGli3-K773/779/784R\u003c/em\u003e) were transfected into HEK293T cells. The relative levels of Gli3R and Gli3FL were analyzed by Western blotting (\u003cstrong\u003eI\u003c/strong\u003e), and their ratio was quantified (\u003cstrong\u003eJ\u003c/strong\u003e). These cells were co-overexpressed with \u003cem\u003eSirt1 \u003c/em\u003e(\u003cstrong\u003eK\u003c/strong\u003e and \u003cstrong\u003eL\u003c/strong\u003e) or treated with Tenovin-1 (100 nM) for 48 hours (\u003cstrong\u003eM\u003c/strong\u003e and \u003cstrong\u003eN\u003c/strong\u003e), the acetylation status of Gli3 was detected by HA immunoprecipitation followed by blotting with anti-acetylated lysine. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. GFP group in panel \u003cstrong\u003eE\u003c/strong\u003e, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 vs. WT group in panel \u003cstrong\u003eJ\u003c/strong\u003e, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ns: not significant.\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7344210/v1/2d250dc2f6371f2878e96f08.jpg"},{"id":89469410,"identity":"9032caf1-c4d1-4219-8bec-3bff14a79f1d","added_by":"auto","created_at":"2025-08-20 09:15:05","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":765730,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSirt1 inhibitors suppress MB cell proliferation \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-B\u003c/strong\u003e \u003cem\u003ePtch1\u003c/em\u003e-deficient primary MB cells were treated with Tenovin-1 and EX-527 at 0, 0.01, 0.05, 0.1, 1, 5, and 10 μM for 48 hours \u003cem\u003ein vitro\u003c/em\u003e, cell proliferation was determined by CCK-8 assay. The IC\u003csub\u003e50\u003c/sub\u003e values of Tenovin-1 (\u003cstrong\u003eA\u003c/strong\u003e) and EX-527 (\u003cstrong\u003eB\u003c/strong\u003e) in inhibiting the proliferation were calculated. \u003cstrong\u003eC-H\u003c/strong\u003e Primary \u003cem\u003ePtch1\u003c/em\u003e-deficient MB cells (\u003cstrong\u003eC\u003c/strong\u003e, \u003cstrong\u003eD\u003c/strong\u003e), human MB cell line DAOY-1 (\u003cstrong\u003eE\u003c/strong\u003e,\u003cstrong\u003e F\u003c/strong\u003e), and \u003cem\u003eSmoA1\u003c/em\u003e-overexpressing MB cell line (\u003cstrong\u003eG\u003c/strong\u003e, \u003cstrong\u003eH\u003c/strong\u003e) were treated with Tenovin-1 or EX-527 (both at 1 μM) or DMSO as control for 48 hours, and were subjected to immunofluorescence staining for Ki67. The percentage of Ki67+ cells/DAPI+ cells was quantified (\u003cstrong\u003eD\u003c/strong\u003e, \u003cstrong\u003eF \u003c/strong\u003eand \u003cstrong\u003eH\u003c/strong\u003e). ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 vs. DMSO group.\u003c/p\u003e","description":"","filename":"Fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7344210/v1/934e78257dfb9d9c375421e2.jpg"},{"id":89471402,"identity":"9aa26be2-d3b6-43c5-95c8-4e3d319d5c7c","added_by":"auto","created_at":"2025-08-20 09:31:06","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":763733,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePharmacological inhibition of Sirt1 attenuates MB progression \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-E\u003c/strong\u003eSubcutaneous xenograft MB model was established in \u003cem\u003eCB17/SCID\u003c/em\u003e mice. Tumor-bearing mice were treated with Tenovin-1 (90 mg/kg) or vehicle control by intraperitoneal injection for 13 consecutive days. n=7. Tumor volume was monitored every day and the fold changes were analyzed (\u003cstrong\u003eA\u003c/strong\u003e). Tumors were dissected for picturing (\u003cstrong\u003eB\u003c/strong\u003e), sectioning for immunohistochemistry staining of Ki67 (\u003cstrong\u003eC\u003c/strong\u003e), evaluation of SHH signaling target genes expression by qPCR (\u003cstrong\u003eD\u003c/strong\u003e) and assessment of Gli3R/Gli3FL protein levels by Western blotting (\u003cstrong\u003eE\u003c/strong\u003e) at the end point of experiments. \u003cstrong\u003eF\u003c/strong\u003e-\u003cstrong\u003eH\u003c/strong\u003ePrimary MB-bearing mice were treated with EX-527 (80 mg/kg/day) by intraperitoneal injection for 2 weeks, tumor tissues were dissected at the end of treatment to prepare frozen sections for immunofluorescence staining of Ki67 (\u003cstrong\u003eF\u003c/strong\u003e), the percentage of Ki67+/DAPI+ cells was quantified (\u003cstrong\u003eG\u003c/strong\u003e), and protein levels of Gli1 and Gli3R/Gli3FL (\u003cstrong\u003eH\u003c/strong\u003e) were assessed by Western blotting. n=3. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7344210/v1/86f2cfb52a191de55ff8a14e.jpg"},{"id":107298213,"identity":"05aa66a9-b468-496e-9fbf-795459560602","added_by":"auto","created_at":"2026-04-20 07:07:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6148075,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7344210/v1/2f4cbb4a-ea3d-45df-a5d1-378cae583b35.pdf"},{"id":89470050,"identity":"fda7dff4-4610-4d1b-826e-5d5efd3075ba","added_by":"auto","created_at":"2025-08-20 09:23:05","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":727222,"visible":true,"origin":"","legend":"Supplementary material","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-7344210/v1/9c6cd4bededbd568ae54d6e8.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"Sirt1 sustains sonic hedgehog signaling to promote medulloblastoma progression through regulating Gli3 processing","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMedulloblastoma (MB) is the most common primary brain malignancy affecting predominantly on children under 14 years of age, and accounts for approximately one-third of all pediatric brain tumors (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). MB almost exclusively occurs in the cerebellum and is characterized by rapid tumor growth and a high propensity for metastasis with high mortality, poor prognosis, and a significant recurrence rate. Currently, the primary treatment strategy involves a combination of surgery, radiotherapy, and chemotherapy (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), however, this approach demonstrates limited efficacy with severe side effects (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Based on distinct molecular and pathological features, MB was traditionally classified into four molecular subtypes: WNT, SHH, Group 3, and Group 4, which are updated into WNT, SHH /TP53 wild-type, SHH/TP53 mutant, and non-WNT/non-SHH type recently (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Among these, SHH-type MB, which results from aberrant activation of the Sonic Hedgehog (SHH) signaling pathway, constitutes approximately 30% of all diagnosed cases and, together with Group 3-MB, represent clinically refractory subtypes (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSHH signaling pathway plays a pivotal role in organismal development, cell proliferation, and tissue differentiation (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Activation of the canonical SHH signaling pathway is triggered by the binding of SHH ligand to its antagonistic receptor Patched 1 (Ptch1), which leads to the release of the transmembrane protein Smoothened (Smo). Thus, Smo is activated and facilitates the nuclear translocation of the Gli (Glioma-associated homologue) transcription factors family from the cytoplasm, thereby initiates the transcription of SHH signaling pathway target genes, including \u003cem\u003eCyclin D1\u003c/em\u003e, \u003cem\u003eN-Myc\u003c/em\u003e, \u003cem\u003ePtch1\u003c/em\u003e, \u003cem\u003ePtch2\u003c/em\u003e, \u003cem\u003eSfrp1\u003c/em\u003e, and \u003cem\u003eGli\u003c/em\u003e itself (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). The Gli family in mammals consists of three members: Gli1, Gli2, and Gli3. Gli1 and Gli2 primarily function as transcriptional activators that positively regulate SHH signaling. In contrast, Gli3 contains a proteolytic processing determinant domain (PDD), which leads to the ubiquitination and proteolytic cleavage of full-length Gli3 protein (Gli3FL) following post-translational modifications such as acetylation and phosphorylation, consequently resulting in the formation of its repressor form (Gli3R, the N-terminal truncated fragment of Gli3FL). Gli3R competes with Gli1/2 for binding to target gene promoters but lacks transcriptional activation capability, thereby suppressing downstream gene expression, functionally, acts as a negative feedback on SHH signaling (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Gli3R inhibited the proliferation of SHH-type MB via such negative feedback mechanism (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). The SHH signaling pathway is also implicated in the pathogenesis of various cancers, including multiple myeloma, colon cancer, pancreatic cancer, basal cell carcinoma, and prostate cancer, as well as SHH-type MB (\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Therefore, targeting this pathway may represent an important anti-tumor therapy approach. Current available SHH pathway inhibitors predominantly target to Smo protein, which frequently acquires resistance-conferring mutations upon treatment, leading to drug resistance and high recurrence rates (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). This necessitates the development of novel therapeutic strategies targeting SHH signaling pathway to overcome the existing treatment limitations.\u003c/p\u003e\u003cp\u003eSHH-type MB originates from cerebellar granule neuron precursors (GNPs) (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). During early cerebellar development, active SHH signaling drives GNP expansion. As cerebellar development progresses, SHH signaling in GNPs gradually diminishes, GNPs cease proliferation and subsequently differentiate into mature granule neurons (GNs) (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Aberrant activation of SHH pathway leads to the sustained and excessive proliferation of GNPs that ultimately drives SHH-type MB tumorigenesis. This pathological activation typically attributes to genetic alterations in SHH pathway components. For instance, SHH-type MB patients have shown \u003cem\u003ePtch1\u003c/em\u003e deletions or inactivating mutations (~\u0026thinsp;43%), \u003cem\u003eSmo\u003c/em\u003e activating mutations (9%), or copy number gains of \u003cem\u003eGli1/2\u003c/em\u003e (9%) (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). The currently available Smo inhibitors are only able to treat MB with the upstream SHH pathway mutations, targeting downstream signaling molecules of the SHH pathway would offer a broader clinical applicability for SHH-type MB and other cancers mediated by this pathway.\u003c/p\u003e\u003cp\u003eSirt1, a member of the seven-protein Sirtuins family, is a class III HDAC. Given the discovery of non-histone protein deacetylation, HDACs have recently been reclassified as lysine deacetylases (KDACs) (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Sirtuins regulate gene expression and participate in diverse cellular processes, including DNA repair, metabolism, oxidative stress response, mitochondrial function, and biogenesis. Dysregulation of their expression or function may contribute to various human diseases, such as cancer, neurodegenerative diseases, metabolic disorders, and cardiovascular diseases (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Among the Sirtuins family, Sirt1 is the most extensively studied member and can deacetylate numerous histone and non-histone proteins (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Studies have demonstrated that Sirt1 is highly expressed in multiple human cancer cell lines and tumor tissues, such as prostate cancer, acute myeloid leukemia, and primary colon cancer (\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). It is interesting to note that expression of Sirt1 was reported to be elevated in human MB cell lines, and its knockdown induces cell cycle arrest \u003cem\u003ein vitro\u003c/em\u003e (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). While, another study demonstrated that the BCL6/BCOR/Sirt1 complex suppresses MB by inhibiting SHH signaling and promoting neurogenesis (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Moreover, emerging information have implicated the involvement of Sirt1 in SHH signaling regulation, for instance, Sirt1 was shown to mediate resveratrol-induced SHH pathway activation (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e); It was also shown to contribute to SHH-dependent microglial activation (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e); Sirt1 may enhance SHH pathway activation in multiple myeloma by deacetylating Gli2 (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Notably, in our previous study, Sirtuins inhibitors showed anti-proliferation effects on MB cells \u003cem\u003ein vitro\u003c/em\u003e (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). In this report, we explore the role of Sirt1 on regulating of SHH signaling and its underlined molecular events associated with cell proliferation and tumor progression of SHH-type MB.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eAnimals\u003c/h2\u003e\u003cp\u003eWild-type (WT) \u003cem\u003eC57BL/6\u003c/em\u003e mice and \u003cem\u003eCB17/SCID\u003c/em\u003e mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. \u003cem\u003eMath1-Cre/Ptch1\u003c/em\u003e\u003csup\u003e\u003cem\u003eloxp/loxp\u003c/em\u003e\u003c/sup\u003e mice were bred in our laboratory as described previously (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). In these mice, \u003cem\u003ePtch1\u003c/em\u003e is conditionally knocked out in Math1-positive cells, which are MB original cells, GNPs. These mice can spontaneously develop MB starting from 4\u0026ndash;6 weeks old. Mice with mixed genders were used in our study. All mice were housed in a specific pathogen-free facility at Soochow University. All animal experiments were approved by and conducted in accordance with the Ethical and Welfare Committee of Soochow University.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eHEK293T and NIH3T3 cells were obtained from Cell Bank/Stem Cell Bank, Chinese Academy of Sciences. Mouse embryonic fibroblasts (MEFs), murine MB cell line and DaoY cells were kindly provided by Dr. Zeng-jie Yang. \u003cem\u003eSmoA1/Gli1/Gli2\u003c/em\u003e stably overexpressed MEF cell lines (\u003cem\u003eSmoA1/Gli1/Gli2\u003c/em\u003e-MEF) were constructed by lentivirus transfection in our lab. DaoY cells were maintained in Minimum Essential Medium (MEM, BasalMedia Technologies, Shanghai, China). All other cells listed above were cultured in DMEM (HyClone, Logan, Utah, USA) containing 10% FBS (Gibco, Grand Island, New York, USA) and 1% penicillin/streptomycin (Beyotime, Shanghai, China).\u003c/p\u003e\u003cp\u003eFor primary GNP and MB cell isolation and culture, we followed a previously described protocol (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Briefly, cerebella or MB tumor tissues were dissected from WT mice at postnatal day 7 (P7) or tumor-bearing \u003cem\u003eMath1-Cre/Ptch1\u003c/em\u003e\u003csup\u003e\u003cem\u003eloxp/loxp\u003c/em\u003e\u003c/sup\u003e mice, respectively, tissues were digested in a buffer containing 250 U/mL DNase (Sigma, St. Louis, Missouri, USA), 10 U/mL papain (Worthington, Lakewood, New Jersey, USA) and 200 \u0026micro;g/mL L-cysteine (Sigma) for 30 minutes at 37\u0026deg;C to acquire a single-cell suspension. The suspension was then centrifuged through a 35\u0026ndash;65% Percoll gradient (Sigma). Cells from the 35\u0026ndash;65% interface were collected and cultured in NB-B27 (Neurobasal with B27 supplement, 2 mM L-glutamine, 1 mM sodium pyruvate, and 1% penicillin/streptomycin).\u003c/p\u003e\n\u003ch3\u003eChemical and protein reagents\u003c/h3\u003e\n\u003cp\u003eRecombinant N terminal of SHH protein (SHH-N) was purchased from Novoprotein (Beijing, China). In certain experiments, NIH3T3 cells were treated with 3 \u0026micro;g/mL SHH-N for indicated hours. Sirt1 inhibitors, Tenovin-1 and EX-527 (both from Selleck, Houston, Texas, USA), were used at indicated doses for cell treatment \u003cem\u003ein vitro\u003c/em\u003e. For treatment of tumor-bearing mice \u003cem\u003ein vivo\u003c/em\u003e, Tenovin-1 (90 mg/kg) and EX-527 (80 mg/kg) was administered by intraperitoneal injections.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry and Immunofluorescent staining\u003c/h3\u003e\n\u003cp\u003eFor immunohistochemistry, normal cerebella or subcutaneous allografts were dissected and then fixed in 4% PFA for 24 hours at 4\u0026deg;C. The preparation and immunohistochemistry staining of paraffin sections were performed by Servicebio Technology (Wuhan, China). Primary antibodies used for immunohistochemistry include anti-Sirt1 (1:200, Santa Cruz, Dallas, Texas, USA) and anti-Ki67 (1:200, Abcam, Waltham, Massachusetts).\u003c/p\u003e\u003cp\u003eFor immunofluorescent staining, tissues were harvested and frozen in Tissue Tek-OCT (Thermo Fisher Scientific, Waltham, Massachusetts), and then sectioned at 10\u0026ndash;12 \u0026micro;m thickness. Sections were blocked in blocking buffer, PBS containing 0.2% Triton X-100 (Biosharp, Hefei, China) and 1% bovine serum albumin (BSA, Fisher bioreagents, Waltham, Massachusetts, USA), for 1 hour at room temperature, and incubated with primary antibodies overnight at 4 \u0026ordm;C. Then, sections were exposed to fluorescein-conjugated secondary antibodies for 2 hours at room temperature on next day. DAPI (Beyotime) was used to counter-stain nuclei for 10 minutes. Cell immunofluorescent staining followed the same protocol. The primary antibodies include anti-Ki67 (1:500), anti-Sirt1 (1:200), anti-HA (1:1000, Proteintech, Wuhan, China) and anti-Tuj1 (1:500, Abcam). The secondary antibodies include Alexa Fluor 488-goat anti-rabbit IgG (1:500, Proteintech), Alexa Fluor 488-goat anti-mouse IgG (1:500, Proteintech), Alexa Fluor 594-goat anti-mouse IgG (1:500, Proteintech) and Alexa Fluor 594-goat anti-rabbit IgG (1:500, Proteintech).\u003c/p\u003e\n\u003ch3\u003eWestern blotting and immunoprecipitation\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eWestern blotting and immunoprecipitation\u003c/div\u003e\u003cp\u003eTissue homogenate or cell lysate was prepared in RIPA buffer (Thermo Fisher Scientific) containing protease and phosphatase inhibitor cocktail (Beyotime), and equal amounts of protein samples were loaded and separated by SDS-PAGE and subsequently transferred onto PVDF membranes (Millipore, Burlington, Massachusetts, USA). The membranes were blocked with blocking buffer (TBS containing 0.05% Tween-20 and 5% defatted milk powder) and incubated overnight at 4\u0026deg;C with primary antibodies against Gli1 (1:1000, Cell Signaling Technology, CST, Danvers, Massachusetts, USA), SHH (1:1000, CST), GAPDH (1:1000, CST), Sirt1 (1:500), Gli3 (1:1000, R\u0026amp;D Systems, Minneapolis, Minnesota, USA), HA (1:3000) and acetylated-Lysine (1:1000, CST). Then the membranes were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibodies, anti-rabbit IgG (1:5000, CST) and anti-mouse IgG (1:5000, CST), at room temperature for 2 hours on the following day. Protein bands were visualized using ECL substrate (Beyotime) and exposed on ChemiScope 3300 mini (Clinx, Suzhou, China).\u003c/p\u003e\u003cp\u003eFor immunoprecipitation, cell lysate was incubated with anti-HA antibody (1:300) overnight at 4\u0026deg;C, and the mixture was incubated with Protein A/G beads (Genscript, Nanjing, China) for another 2 hours at 4\u0026deg;C on next day. The beads were washed with PBS for 3\u0026ndash;5 times and resuspended in SDS loading buffer for Western blotting analysis.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eReal-time quantitative PCR (qPCR)\u003c/h2\u003e\u003cp\u003eTotal RNA was isolated from tissue homogenate or cell lysate using TRIzol reagent (Vazyme Biotech, Nanjing, China) in RNase-free conditions. cDNA was synthesized using RNA reverse transcription kit (Vazyme Biotech). Then, qPCR reactions were performed using SYBR qPCR Master Mix (Vazyme Biotech).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eGenetic manipulation\u003c/h3\u003e\n\u003cp\u003eTo knock down \u003cem\u003eSirt1\u003c/em\u003e in MB and MEF cells, lentiviruses carrying shRNAs constructed in pLV[shRNA]-mCherry-U6 plasmid were produced following regular protocol in HEK293T cells, and the supernatant in the relevant medium was collected for cell infection.\u003c/p\u003e\u003cp\u003eFor gene overexpression in cells, murine \u003cem\u003eSmoA1\u003c/em\u003e (\u003cem\u003eSmo W539L\u003c/em\u003e, activate mutation of Smo); \u003cem\u003eGli1\u003c/em\u003e; \u003cem\u003eGli2\u003c/em\u003e and \u003cem\u003eSirt1\u003c/em\u003e were constructed into vector pLV-AcGFP; \u003cem\u003eGli3\u003c/em\u003e (full length, WT or K773R; K779R; K784R and K773/779/784R mutants) and \u003cem\u003eGli3R\u003c/em\u003e were constructed into vector pLV-HA. Lentiviruses carrying indicated genes listed above were produced to infect primary MB, MEF, NIH3T3 and HEK293T cells.\u003c/p\u003e\n\u003ch3\u003eCell counting kit-8 (CCK-8) assay\u003c/h3\u003e\n\u003cp\u003eCCK-8 assay was performed to detect cell viability. Cells were inoculated into 96-well plates at 3 ⅹ10\u003csup\u003e3\u003c/sup\u003e/ well density and cultured with indicated treatment for 48 hours. Then cells were incubated with 10 \u0026micro;L of CCK-8 reagent (Beyotime) for 1 hour to detect optical density at 450 nm wave length.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eOrthotopic and subcutaneous transplantation MB models\u003c/h2\u003e\u003cp\u003eFour- to five-week-old \u003cem\u003eCB17/SCID\u003c/em\u003e mice were used for both models. For orthotopic transplantation model, primary MB cells were infected with a lentivirus carrying \u003cem\u003eSirt1\u003c/em\u003e ShRNA or scrambled control, then mCherry\u0026thinsp;+\u0026thinsp;cells were sorted by flow cytometry 24 hours later, and 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells suspended in 5 \u0026micro;l PBS per mouse were intracranially injected into mice cerebellar.\u003c/p\u003e\u003cp\u003eFor subcutaneous transplantation model, mice were injected with 2\u0026times;10\u003csup\u003e6\u003c/sup\u003e primary MB cells per mouse in the hind flanks subcutaneously. Tumor volume was measured and calculated as 1/2\u0026times; (W\u003csup\u003e2\u003c/sup\u003e\u0026times;L) (W, width; L, length). When the tumor volume reached 200 mm\u003csup\u003e3\u003c/sup\u003e, the mice were randomly grouped and received treatments. The sirt1 inhibitor, Tenovin-1 (90 mg/kg bodyweight), or vehicle control was injected intraperitoneally into tumor-bearing mice daily at designated sites until the tumor size in the control group reached 1 cm\u003csup\u003e3\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eDatabase analysis\u003c/h2\u003e\u003cp\u003eRelevant MB patient data were acquired from R2 database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://hgserver1.amc.nl/cgi-bin/r2/main.cgi\u003c/span\u003e\u003cspan address=\"https://hgserver1.amc.nl/cgi-bin/r2/main.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Single-cell RNA sequencing data of \u003cem\u003eSmoM2\u003c/em\u003e-driven SHH-type MB was obtained from an open database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gershon-lab.med.unc.edu/single-cell/\u003c/span\u003e\u003cspan address=\"http://gershon-lab.med.unc.edu/single-cell/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eStatistics\u003c/h2\u003e\u003cp\u003eExperimental data were analyzed using GraphPad Prism software 9.5. Unpaired \u003cem\u003eStudent\u0026rsquo;s t\u003c/em\u003e test was used to calculate the difference in experiments containing two groups when data exhibit Gaussian distribution. Otherwise, data was analyzed via a non-parametric \u003cem\u003eStudent\u0026rsquo;s t\u003c/em\u003e test. For experiments containing three or more groups, one-way or two-way ANOVA was used to determine the levels of difference. Data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to be significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eSirt1 expression is upregulated in SHH-type MB and positively correlates with SHH signaling and MB proliferation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn our previous study, epigenetic compounds have been screened targeting MB (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e), we further identified Sirtuins inhibitors significantly suppressed MB cell proliferation \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To further investigate the functional role of Sirtuins in SHH signaling and MB tumorigenesis, we first detected the expression patterns of Sirtuin family members in primary MB cells and GNPs, the internal granular layer of normal adult mouse cerebellum served as the mature GNs control. Quantitative mRNA analysis revealed that \u003cem\u003eSirt1\u003c/em\u003e was significantly highly expressed in MB cells and GNPs compared to GNs with the highest expression in MB cells (MB\u0026thinsp;\u0026gt;\u0026thinsp;GNPs\u0026thinsp;\u0026gt;\u0026thinsp;GNs); \u003cem\u003eSirt5\u003c/em\u003e displayed a similar pattern but with much weaker expression; Remained other subfamilies of Sirtuins showed no significant changes across these cell types (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Meanwhile, detection of the protein expression by either immunohistochemistry or Western blotting confirmed the significant elevated expression of Sirt1 in tumor region compared to adjacent normal cerebellum (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), so was in tumor cells versus GNPs/GNs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-E). It is known that MB cells exhibit hyperproliferative characteristics, GNPs maintain active proliferation, while GNs represent the terminally differentiated cells. We found that the activation status of the SHH signaling (as indicated by Gli1 protein levels) followed a consistent gradient: MB\u0026thinsp;\u0026gt;\u0026thinsp;GNPs\u0026thinsp;\u0026gt;\u0026thinsp;GNs, which were, intriguingly, in according with Sirt1 expressing pattern among these cell types, suggesting a potential role of Sirt1 in regulation of proliferation and SHH pathway activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Since it has been demonstrated that cultured MB cells exhibited progressive SHH pathway inactivation due to the lack of tumor microenvironment (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e), we further showed the time-dependent SHH pathway inactivation in the cultured MB cells as evidenced by significant downregulation of SHH target genes (\u003cem\u003eGli1\u003c/em\u003e, \u003cem\u003eGli2\u003c/em\u003e, \u003cem\u003ePtch2\u003c/em\u003e, and \u003cem\u003eSfrp1\u003c/em\u003e), and the concomitant decreased \u003cem\u003eSirt1\u003c/em\u003e expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), which was in parallelled with the gradually ceased proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG-H), revealing a positive correlation between \u003cem\u003eSirt1\u003c/em\u003e expression and cellular proliferation/SHH signaling activity. To exclude the possibility that this correlation may be specific to \u003cem\u003ePtch1\u003c/em\u003e-deficient tumors, we analyzed single-cell RNA sequencing data from \u003cem\u003eSmoM2\u003c/em\u003e (active mutant of \u003cem\u003eSmo\u003c/em\u003e)-driven MB mouse model. We observed that \u003cem\u003eSirt1\u003c/em\u003e expressing population was predominantly localized to SHH-activated (Gli1+) cell clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). To further validate whether this \u003cem\u003eSirt1\u003c/em\u003e expression pattern is clinically relevant, we analyzed \u003cem\u003eSirt1\u003c/em\u003e mRNA levels in tumor tissues from patients across different MB subgroups via R2 database. Notably, \u003cem\u003eSirt1\u003c/em\u003e expression was significantly elevated in SHH-type MB compared to other molecular subtypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ). These findings demonstrated that Sirt1 is preferentially overexpressed in SHH-driven MB, and its expression positively correlates with both proliferative status and SHH pathway activity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSirt1\u003c/b\u003e \u003cb\u003eknockdown suppresses SHH signaling and MB cell proliferation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo verify whether Sirt1 is involved in the regulation of MB proliferation, we first knocked down \u003cem\u003eSirt1\u003c/em\u003e in primary MB cells and detect the tumor proliferation marker Ki67 by immunofluorescence staining. Compared with the scrambled control, the percentage of Ki67\u0026thinsp;+\u0026thinsp;cells was significantly reduced in \u003cem\u003eSirt1\u003c/em\u003e-knocked-down MB cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). Meanwhile, \u003cem\u003eSirt1\u003c/em\u003e knockdown markedly suppressed the expression of SHH pathway target genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) and Gli1 protein expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), indicating the inhibition of the SHH signaling. We next injected \u003cem\u003eSirt1\u003c/em\u003e-knocked-down MB cells into the cerebellum of \u003cem\u003eCB17/SCID\u003c/em\u003e mice to evaluate the effect of \u003cem\u003eSirt1\u003c/em\u003e defect in an orthotopic MB xenograft model. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-F, \u003cem\u003eSirt1\u003c/em\u003e knockdown significantly inhibited MB formation and prolonged the survival of the experimental mice, confirming the importance of Sirt1 in SHH signaling and MB proliferation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNIH3T3 cells possess all key components of the SHH signaling pathway and can be activated by exogenous SHH ligands, making them a widely used model for studying SHH pathway regulation and mechanisms, we thus employed this cell line to investigate whether Sirt1 directly activates SHH signal transduction. NIH3T3 cells were stably infected with lentiviruses encoding either \u003cem\u003eSirt1\u003c/em\u003e shRNA or \u003cem\u003eSirt1\u003c/em\u003e cDNA. The transfecting efficacy was confirmed by Western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). After treatment with recombinant SHH-N protein (N terminal of SHH ligand possessing bioactivity, 3 \u0026micro;g/mL), increased Gli1 levels was detected in WT NIH3T3 cells, confirming SHH pathway activation. While, \u003cem\u003eSirt1\u003c/em\u003e-knockdown cells showed reduced Gli1 expression, revealing SHH-N stimulated SHH pathway activation was associated with \u003cem\u003eSirt1\u003c/em\u003e expression. In support, \u003cem\u003eSirt1\u003c/em\u003e-overexpressing cells exhibited the elevated Gli1 levels, which was further enhanced upon SHH-N treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH-I).\u003c/p\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eSirt1 regulates SHH signaling downstream of Gli1/Gli2 by modulating Gli3 processing\u003c/h2\u003e\u003cp\u003eActivation of canonical SHH signaling pathway involves a cascade of sequential key signaling molecules: SHH ligand‒Ptch‒Smo‒Gli1/2. We already identified that Sirt1 functions downstream of SHH ligand and Ptch1 since NIH3T3 cells were under SHH ligand stimulation and primary MB cells carried \u003cem\u003ePtch1\u003c/em\u003e gene deletion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). To further define Sirt1's action in the signaling cascade, we established three distinct SHH pathway-activated MEF cell lines by using a lentivirus-mediated stable overexpression system: \u003cem\u003eSmoA1\u003c/em\u003e-MEF (active \u003cem\u003eSmo\u003c/em\u003e mutant, \u003cem\u003eW539L\u003c/em\u003e), \u003cem\u003eGli1\u003c/em\u003e-MEF, and \u003cem\u003eGli2\u003c/em\u003e-MEF. The efficiency of overexpression was confirmed by qPCR, and each of these genetic modifications effectively activated the SHH pathway independently (Supplementary Fig.\u0026nbsp;1). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-D, \u003cem\u003eSirt1\u003c/em\u003e knockdown in all three mutants as well as in WT MEF cells consistently downregulated SHH pathway target genes as compared to scrambled control. These results indicate that Sirt1 regulates SHH signaling cascades downstream of Gli1/2.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eGli3FL undergoes processing to generate Gli3R, which negatively regulates the SHH pathway through competing with Gli1/Gli2. To investigate whether Sirt1 regulates this pathway by modulating Gli3 processing, we examined the protein levels of Gli3R and Gli3FL, as well as their ratio (Gli3R/Gli3FL) in NIH3T3 cells with either \u003cem\u003eSirt1\u003c/em\u003e knockdown or overexpression. This ratio is considered to be an indicator of the extent of Gli3 processing. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-F, in the absence of SHH ligand stimulation, no significant differences in Gli3 processing were observed among the experimental groups; In contrast, in the presence of SHH ligand stimulation, \u003cem\u003eSirt1\u003c/em\u003e-knockdown cells exhibited a significant increase in the Gli3R/Gli3FL ratio, and as expected, \u003cem\u003eSirt1\u003c/em\u003e-overexpressing cells decreased the ratio, clearly demonstrating that Sirt1 suppressed the proteolytic processing of Gli3. Similarly, \u003cem\u003eSirt1\u003c/em\u003e-knockdown primary MB cells as well as in the orthotopic xenograft showed significantly elevated Gli3R/Gli3FL ratios (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-H). To confirm whether Sirt1 maintains SHH pathway activity by inhibiting Gli3 processing, we performed rescue experiments in MEF cells. The results showed that \u003cem\u003eSirt1\u003c/em\u003e overexpression alone was sufficient to activate SHH signaling, while co-expression of Gli3R abolished the Sirt-mediated SHH pathway activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eSirt1 directly interacts with and deacetylates Gli3\u003c/h2\u003e\u003cp\u003eIt was reported that Sirt1 directly interacts with Gli2 and mediates its deacetylation (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Given the high homology between Gli2 and Gli3, we hypothesized that Sirt1 might similarly interact with and deacetylate Gli3. Due to the lack of reliable antibodies to immunoprecipitate endogenous Gli3 or Sirt1, we employed NIH3T3 cells transfected with either HA-tagged Gli3FL or HA-Gli3R. Co-IP experiments using anti-HA antibody demonstrated that endogenous Sirt1 specifically co-precipitated with Gli3FL, but not with Gli3R (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The observation was confirmed by immunofluorescence staining which revealed the colocalization between endogenous Sirt1 and exogenous HA-Gli3FL (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In cellular fractionation separations, Sirt1 was present in both nuclear and cytoplasmic compartments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Given that Gli3FL predominantly localizes to the cytoplasm that is translocated in to the nucleus when processed to Gli3R, the observed cytoplasmic colocalization of Sirt1 with Gli3FL is in agreement with co-IP results. Given Sirt1 might itself be a direct target of SHH pathway (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e), to eliminate potential confounding effects of SHH signaling on Sirt1 protein levels, we utilized HEK293T cells, which exhibit minimal basal SHH pathway activity, to establish a HA-Gli3FL stably expressing line. Sirt1 overexpression significantly suppressed Gli3 processing; while treatment with Sirt1 inhibitor Tenovin-1 (100 nM) promoted it (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-E). We next assessed Gli3 acetylation status by co-IP, which revealed that Gli3 underwent endogenous acetylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF-G), and Tenovin-1 treatment increased Gli3 acetylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG), suggesting that Sirt1 indeed suppressed Gli3 acetylation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further investigate the molecular mechanism by which Sirt1 regulates Gli3 processing, we analyzed the lysine acetyltransferase (KAT)-specific acetylation site database (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e) and the homologous acetylation sites of Gli2. We predicted three potential acetylation sites on the Gli3 protein: K773, K770, and K784 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Plasmids for each or all three of those lysine mutants (replaced by arginine) were prepared and transfected to HEK293T cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI-J, all single-site mutations reduced the Gli3R/Gli3FL ratio, while simultaneous mutation of all three sites led to a more dramatic decrease, indicating that these acetylation sites are critical for Gli3FL processing. To determine whether Sirt1 regulates Gli3 acetylation through these three sites, we overexpressed \u003cem\u003eSirt1\u003c/em\u003e or applied its inhibitor in WT \u003cem\u003eHA-Gli3FL\u003c/em\u003e- and mutant \u003cem\u003eHA-Gli3FL (K773/779/784R)\u003c/em\u003e-cells, respectively. The results showed that \u003cem\u003eSirt1\u003c/em\u003e overexpression suppressed Gli3 acetylation, while Sirt1 inhibitor treatment increased Gli3 acetylation in WT \u003cem\u003eGli3\u003c/em\u003e overexpressing cells. In contrast, mutant Gli3 did not alter acetylation by \u003cem\u003eSirt1\u003c/em\u003e overexpression or Sirt1 inhibition. We also observed that the acetylation levels of the K773/779/784R-mutated Gli3 were lower than those of the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK-N). Taken altogether, our data indicate that Sirt1 deacetylates Gli3 at K773, K779, and K784 residues in the C-terminal transcriptional activation domain.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSirt1 inhibitors suppress MB cell proliferation\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSo far, we have revealed that Sirt1 regulates the SHH signaling pathway by interacting with Gli3. We wondered whether selective pharmacological inhibitors of Sirt1 could suppress the proliferation of SHH-type MB. We selected two well-characterized Sirt1 inhibitors, Tenovin-1 and EX-527, and treated \u003cem\u003eMath1-cre/Ptch1\u003c/em\u003e\u003csup\u003e\u003cem\u003eloxp/loxp\u003c/em\u003e\u003c/sup\u003e mice derived primary MB cells, cell viability exhibited that both inhibitors suppressed MB cell proliferation in a dose-dependent manner, with IC\u003csub\u003e50\u003c/sub\u003e values of 241.3 nM for Tenovin-1 and 1.2 \u0026micro;M for EX-527 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B). Immunofluorescence staining revealed that Tenovin-1(1\u0026micro;M) or EX-527 (1\u0026micro;M) treatment significantly reduced the percentage of Ki67\u0026thinsp;+\u0026thinsp;cells, while increasing the proportion of differentiated (Tuj1+) cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-D). We further confirmed the anti-proliferative effects of these inhibitors in human SHH-MB cells (DAOY-1) and \u003cem\u003eSmoA1\u003c/em\u003e-overexpressing MB cell line. The \u003cem\u003eSmoA1\u003c/em\u003e mutation confers resistance to the Smo inhibitor Vismodegib (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Consistent with the results in \u003cem\u003ePtch1\u003c/em\u003e-deficient mouse MB cells, Tenovin-1 or EX-527 effectively suppressed proliferation in both DAOY-1 and Vismodegib-resistant \u003cem\u003eSmoA1\u003c/em\u003e-MB cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-H).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ePharmacological inhibition of Sirt1 attenuates MB progression\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe next assessed the therapeutic potential of Sirt1 inhibitors \u003cem\u003ein vivo\u003c/em\u003e. Since Tenovin-1 has limited blood-brain barrier permeability, we employed a subcutaneous MB xenograft approach to evaluate its efficacy. Treatment with Tenovin-1 (90 mg/kg/day, administered via intraperitoneal injection) for 2 weeks markedly inhibited tumor growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B). Immunohistochemical analysis of tumor tissues confirmed that Tenovin-1 treatment significantly reduced Ki67\u0026thinsp;+\u0026thinsp;cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). As expected, Tenovin-1 treatment also suppressed SHH pathway target genes expression, but increased Gli3R/Gli3FL ratio in tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-E).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe also tested EX-527, a highly specific Sirt1 inhibitor with demonstrated blood-brain barrier permeability (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e), on primary MB in \u003cem\u003eMath1-cre/Ptch1\u003c/em\u003e\u003csup\u003e\u003cem\u003eloxp/loxp\u003c/em\u003e\u003c/sup\u003e mice. We treated these mice with EX-527 (80 mg/kg/day via intraperitoneal injection), started from week 4 for 14 consecutive days. Immunofluorescence staining showed significantly reduced tumor cell proliferation (Ki67\u0026thinsp;+\u0026thinsp;cells) in EX-527-treated mice compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF-G). Western blotting analysis demonstrated attenuated Gli1 expression and enhanced Gli3 processing in primary tumors with EX-527 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH).\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlthough significant progress has been made in elucidating the pathogenesis of MB, no significant clinical breakthroughs have been achieved (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Therefore, there is an urgent need for more comprehensive investigations of MB pathogenesis mechanisms to development new therapeutic targets. In current study, we investigate the role and the underlying mechanism of Sirt1 in the SHH signaling pathway and SHH-type MB. We found that Sirt1 was highly expressed in both human and mouse SHH-type MB, and its expression positively correlated with SHH pathway activity and cell proliferation in MB cells. To investigate its role in MB, we knocked down \u003cem\u003eSirt1\u003c/em\u003e in primary MB cells and observed a significant inhibition of MB proliferation \u003cem\u003ein vitro\u003c/em\u003e and a reduction in SHH pathway activity. Moreover, MB cells with \u003cem\u003eSirt1\u003c/em\u003e defect almost lost their tumor-forming ability when orthotopically transplanted into mouse cerebellum. These data indicate that Sirt1 positively regulates SHH signaling and MB proliferation both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. We further validated our findings using NIH3T3 cells, a tool cell line that possesses all components of the SHH pathway but requires exogenous SHH ligand for pathway activation. Knocking down \u003cem\u003eSirt1\u003c/em\u003e in these cells also suppressed SHH pathway activation, confirming that Sirt1 directly regulates SHH pathway.\u003c/p\u003e\u003cp\u003eTo identify the specific cascade at which Sirt1 modulates the SHH signal pathway, we knocked down \u003cem\u003eSirt1\u003c/em\u003e in MEF cells overexpressing \u003cem\u003eSmoA1\u003c/em\u003e, \u003cem\u003eGli1\u003c/em\u003e, or \u003cem\u003eGli2\u003c/em\u003e, respectively, and found that \u003cem\u003eSirt1\u003c/em\u003e depletion significantly inhibited SHH pathway activation in all cases, suggesting that Sirt1 functions downstream of Gli1/2. Considering that Gli3 always acts as negative feedback of SHH signaling through processing into Gli3R (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e) at the downstream of Gli1/2. Although Gli2 also contains a PDD, its processing into Gli2R is inefficient and typically does not contribute significantly to negative feedback regulation (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Thus, in canonical SHH signaling, the relative abundance of Gli3R versus activated Gli2/Gli3 determines the net pathway activity (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). We then focused on investigating whether Sirt1 affects the generation of Gli3R. Results showed that \u003cem\u003eSirt1\u003c/em\u003e knockdown in NIH3T3 cells significantly enhanced Gli3R production while reduced SHH pathway activity concurrently, whereas \u003cem\u003eSirt1\u003c/em\u003e overexpression suppressed Gli3R generation and upregulated SHH signaling. To further confirm whether Sirt1 regulates the SHH pathway through modulating Gli3 processing, we overexpressed \u003cem\u003eSirt1\u003c/em\u003e with or without co-overexpression of \u003cem\u003eGli3R\u003c/em\u003e in NIH3T3 cells, and found that overexpression of \u003cem\u003eSirt1\u003c/em\u003e alone was sufficient to trigger SHH pathway activation, whereas co-expression of \u003cem\u003eGli3R\u003c/em\u003e dampened the activation, confirming that Sirt1 positively regulates SHH pathway by inhibiting Gli3 processing into Gli3R.\u003c/p\u003e\u003cp\u003eNext, we explored the molecular mechanism by which Sirt1 inhibits Gli3 processing. Gli3 processing involves a multistep process, including acetylation, phosphorylation, and subsequent ubiquitin-proteasome-mediated degradation (\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). This process is tightly regulated by diverse mechanisms (\u003cspan additionalcitationids=\"CR46 CR47\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e), which may significantly influence both physiological and pathological SHH-mediated processes. For example: Cadherin-7 has been shown to enhance SHH signaling during neural tube development by suppressing Gli3 processing (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e); Set7-mediated methylation of Gli3 promotes SHH pathway activation (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Given the deacetylase property of Sirt1, and its action on deacetylating Gli2 (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e), we wondered whether it may interact and deacetylate Gli3 directly. Co-IP experiments revealed that Sirt1 interacts with Gli3FL but not Gli3R, suggesting that Sirt1 may regulate the initial steps of Gli3 processing and that the interaction domain might be lost during Gli3R generation. To exclude the potential influence of SHH signaling on Sirt1 itself, we used SHH-inactive HEK293T cells to study the specific mechanism of Sirt1-mediated Gli3 processing regulation. Overexpression of \u003cem\u003eSirt1\u003c/em\u003e as well as treatment with the Sirt1 enzymatic inhibitor, Tenovin-1, in these cells reproduced the previous observations: \u003cem\u003eSirt1\u003c/em\u003e overexpression inhibited Gli3 processing, while the inhibitor promoted it. We then examined Sirt1\u0026rsquo;s effect on Gli3 acetylation in these cells. We found that overexpressed Gli3 in HEK293T cells underwent acetylation, which was enhanced by Sirt1 inhibition, indicating that Sirt1 suppresses Gli3 acetylation in a SHH pathway-independent manner. To verify whether Gli3 acetylation affects its processing, we mutated key lysine residues in Gli3 that are critical for acetylation, and overexpressed the mutants in HEK293T cells. The mutations drastically reduced Gli3 processing. Furthermore, in cells overexpressing the mutant Gli3, neither \u003cem\u003eSirt1\u003c/em\u003e overexpression nor inhibitor treatment altered Gli3 acetylation levels, confirming that these residues are essential for Sirt1-mediated deacetylation of Gli3.\u003c/p\u003e\u003cp\u003eFinally, we assessed the therapeutic feasibility of targeting Sirt1 in SHH-type MB. We evaluated two well-characterized Sirt1 inhibitors, Tenovin-1 and EX527. \u003cem\u003eIn vitro\u003c/em\u003e studies demonstrated that both inhibitors effectively suppressed proliferation across multiple MB cell models, including primary mouse MB cells, human SHH-type MB cells, and \u003cem\u003eSmoA1\u003c/em\u003e-overexpressing MB cells. Importantly, \u003cem\u003ein vivo\u003c/em\u003e validation using distinct tumor models revealed consistent anti-tumor efficacy: Tenovin-1 treatment in subcutaneous xenografts significantly enhanced Gli3 processing, suppressed SHH pathway activation, and markedly inhibited tumor growth. Similarly, EX-527 administration in a genetically engineered primary MB model faithfully reproduced these anti-proliferative effects. The concordant therapeutic outcomes observed across both transplanted and spontaneous MB models strongly support Sirt1 as a novel and promising therapeutic target for SHH-type MB.\u003c/p\u003e\u003cp\u003eThe striking discovery of the present work revealed the critical role of Sirt1 in SHH signaling and SHH-type MB, and confirmed its action at the downstream of SHH pathway, making it a valuable and promising target for SHH-driven malignancies treatment. Since current clinical approaches predominantly employ Smo inhibitors, such as Vismodegib, as the principal pharmacological strategy. These agents are substantially limited by the rapid emergence of acquired resistance mediated by \u003cem\u003eSmo\u003c/em\u003e mutations, frequently resulting in tumor relapse (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Furthermore, Smo-targeted therapies are only effective against the subset of SHH-MB cases harboring upstream pathway alterations (approximately 50%). While targeting Sirt1 may overcome these limitations, which would potentially offer broader clinical applicability for both SHH-MB and other SHH-driven diseases.\u003c/p\u003e\u003cp\u003eOur present work also elaborated that Sirt1 regulates SHH pathway by disrupting the negative feedback loop mediated by Gli3R. As established, genetic mutations serve as initiating events that activate oncogenic signaling pathways, sustained pathway activation is required to maintain tumor cell proliferation and facilitate the progression to full-grown tumors. In SHH-type MB, for instance, excessive SHH signaling triggered by genetic mutations such as \u003cem\u003ePtch1\u003c/em\u003e deletion induces feedback inhibition through enhanced Gli3 processing into Gli3R. Sirt1 is demonstrated here acting as the \u0026ldquo;second hit\u0026rdquo; to breakdown this negative feedback and ultimately lead to the tumor formation and progression. Mechanistically, out work illustrated that Sirt1 suppresses Gli3 processing via post-translational modifications, which also emphasizes the emerging recognition of post-translational modifications and epigenetic regulation in SHH-type MB pathogenesis.\u003c/p\u003e\u003cp\u003eAdmittedly, the precise mechanisms underlying Sirt1-mediated regulation of the SHH pathway require further in-depth investigation. Several critical questions remain to be addressed: Whether the interaction between Sirt1 and Gli3 depends on specific acetylation sites or distinct binding domains; How the binding affinity between these proteins is regulated; And the exact binding regions and sites need to be systematically mapped using various truncated protein fragments. Furthermore, while Gli1 and Gli2 are predominantly nuclear proteins, Gli3FL mainly localizes to the cytoplasm. Interestingly, aberrant cytoplasmic accumulation of Sirt1 has been linked to enhanced protein stability regulated by PI3K/IGF-1R signaling (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). Does Sirt1 modulate SHH pathway activity by shuttling between the nucleus and cytoplasm? These aspects warrant thorough exploration to fully elucidate the crosstalk between Sirt1 and SHH signaling in MB pathogenesis.\u003c/p\u003e\u003cp\u003eIn summary, our findings support a mechanistic model wherein Sirt1-mediated deacetylation of Gli3 inhibits its proteolytic processing into the repressor form (Gli3R), consequently sustaining SHH pathway activation and promoting SHH-type MB proliferation. This study elucidates a previously unrecognized regulatory axis: Sirt1‒Gli3 processing‒SHH pathway activity, providing novel mechanistic insights into MB pathogenesis. Specifically, we demonstrate that Sirt1 potentiates SHH signaling through suppression of the Gli3R-mediated negative feedback loop, representing a critical post-translational regulatory layer in SHH-driven tumorigenesis. The present data also implicate Sirt1 as a potential target for SHH-type MB drug discovery.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\n\u003cp\u003e All animal experiments were carried out according to guidelines approved by the Institutional Animal Care and Use Committee of Soochow University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003eAuthors' contributions\u003c/h2\u003e\n\u003cp\u003eJH: project management, research conduction, experiments performance, data analysis, results interpretation, and manuscript preparation. CNZ: research conduction, experiments performance, data analysis, and results interpretation. YYF/YHL/PPP/MY/YHQ: experiments performance, data acquisition. LZ/LTZ: data analysis, results interpretation, funding. ZXC: results interpretation, manuscript preparation, funding. YW: project management, research conduction, data analysis, results interpretation, manuscript preparation, funding.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eThis research was supported by National Natural Science Foundation of China (82373900 and 82073873 to Yuan Wang, 82072798 to Li Zhang, 81973334 to Xue-Chu Zhen, 32371016 and 31970909 to Long-Tai Zheng), the National Innovation of Science and Technology-2030 (Program of Brain Science and Brain-Inspired Intelligence Technology) (2021ZD0204004), the National Key Research and Development Program of China (2021YFE0206000) and the Priority Academic Program Development of the Jiangsu Higher Education Institutes (PAPD).\u003c/p\u003e\n\u003ch2\u003eAvailability of data\u003c/h2\u003e\n\u003cp\u003eThe data that support the findings of this study are available from corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNorthcott PA, Robinson GW, Kratz CP, Mabbott DJ, Pomeroy SL, Clifford SC, et al. 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Int J Biol Sci. 2010;6(6):599-612.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"oncogene","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"onc","sideBox":"Learn more about [Oncogene](http://www.nature.com/onc/)","snPcode":"41388","submissionUrl":"https://mts-onc.nature.com/cgi-bin/main.plex","title":"Oncogene","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Medulloblastoma, SHH signaling, Sirt1, Gli3 processing","lastPublishedDoi":"10.21203/rs.3.rs-7344210/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7344210/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMedulloblastoma (MB), the most common malignant pediatric brain tumor with poor prognosis, high recurrence, and severe treatment-related toxicities. One-third of MB are driven by aberrant activation of the Sonic hedgehog (SHH) signaling pathway. In current study, through analysis of clinical patient cohorts and animal model database, and utilizing genetically engineered primary and xenograft mouse MB models, we investigated the role of Sirtuin1 (Sirt1), a class III histone deacetylase (HDAC), in SHH signaling and MB. We found that Sirt1 was highly expressed in both human and mouse SHH-type MB, and its expression positively correlated with SHH pathway activity and tumor proliferation. Knockdown of \u003cem\u003eSirt1\u003c/em\u003e in primary MB cells significantly suppressed SHH signaling and MB proliferation \u003cem\u003ein vitro\u003c/em\u003e, further impaired neoplastic progression and extended survival in orthotopic transplantation MB model. Mechanistically, we discovered that Sirt1 modulates SHH signaling at downstream by interacting with and deacetylating full-length Gli3 (Gli3FL), thereby inhibiting its proteolytic processing into the repressor form (Gli3R), which attenuates the negative feedback regulation of SHH signaling, sustaining pathway activation and promoting tumor progression. Importantly, pharmacological inhibition of Sirt1 demonstrated promising therapeutic efficacy in both subcutaneous transplantation and primary MB models. Our findings identify Sirt1 as a potential therapeutic target for SHH-driven MB and other cancers.\u003c/p\u003e","manuscriptTitle":"Sirt1 sustains sonic hedgehog signaling to promote medulloblastoma progression through regulating Gli3 processing","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-20 09:15:01","doi":"10.21203/rs.3.rs-7344210/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-10-30T14:29:24+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-09-05T10:35:40+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-08-18T18:40:51+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-08-18T08:43:57+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-08-13T02:59:21+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-12T11:24:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-11T08:38:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"Oncogene","date":"2025-08-11T08:38:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"oncogene","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"onc","sideBox":"Learn more about [Oncogene](http://www.nature.com/onc/)","snPcode":"41388","submissionUrl":"https://mts-onc.nature.com/cgi-bin/main.plex","title":"Oncogene","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e403f683-f51b-4e12-b63d-06229935768a","owner":[],"postedDate":"August 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":53075724,"name":"Biological sciences/Cancer/CNS cancer"},{"id":53075725,"name":"Biological sciences/Molecular biology/Post-translational modifications/Acetylation"},{"id":53075726,"name":"Biological sciences/Cell biology/Mechanisms of disease"}],"tags":[],"updatedAt":"2026-04-20T07:07:41+00:00","versionOfRecord":{"articleIdentity":"rs-7344210","link":"https://doi.org/10.1038/s41388-026-03781-1","journal":{"identity":"oncogene","isVorOnly":false,"title":"Oncogene"},"publishedOn":"2026-04-18 04:00:00","publishedOnDateReadable":"April 18th, 2026"},"versionCreatedAt":"2025-08-20 09:15:01","video":"","vorDoi":"10.1038/s41388-026-03781-1","vorDoiUrl":"https://doi.org/10.1038/s41388-026-03781-1","workflowStages":[]},"version":"v1","identity":"rs-7344210","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7344210","identity":"rs-7344210","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}