A selective Fibroblast Growth Factor Receptor 1/2 PROTAC degrader with antitumor activity.

The aberrant activation of fibroblast growth factor receptor (FGFR) acts as a potent driver of multiple types of human cancers. Despite the development of several conventional small-molecular FGFR inhibitors, their clinical efficacy is largely compromised due to low selectivity and side effects. Here, we report the selective FGFR1/2-targeting proteolysis targeting chimeric (PROTAC), BR-cpd7 that displays significant isoform specificity to FGFR1/2 with DC50 values around 10 nM, while sparing FGFR3. The following mechanistic investigation reveals the reduced FGFR signaling, through which BR-cpd7 induces cell cycle arrest and consequently blocks the proliferation of multiple FGFR1/2-dependent tumor cells. Importantly, BR-cpd7 has almost no anti-proliferative activity against cancer cells without FGFR aberrations, furtherly supporting its selectivity. In vivo, BR-cpd7 exhibits robust antitumor effects in FGFR1-dependent lung cancer at well-tolerated dose schedules, accompanied by complete FGFR1 depletion. Overall, we identify BR-cpd7 as a promising candidate for developing a selective FGFR1/2-targeted agent, thereby offering a new therapeutic strategy for human cancers in which FGFR1/2 plays a critical role.

Apigenin Suppresses Innate Immune Responses and Ameliorates Lipopolysaccharide-Induced Inflammation via Inhibition of STING/IRF3 Pathway.

The stimulator of interferon genes (STING) signaling pathway is crucial for the pathogenesis of autoimmune and inflammatory disorders, including acute lung injury (ALI). Apigenin (4[Formula: see text],5,7-trihydroxyflavone) is a natural flavonoid widely found in fruits, vegetables, and Chinese medicinal herbs that exhibits a range of pharmacological effects, such as antibacterial and anti-inflammatory activities. However, the efficacy of apigenin in STING pathway-mediated diseases remains unclear. Accordingly, this study screened Chinese medicines to identify potent agents that reduced the synthesis of type I interferons (IFNs). The results revealed apigenin as a potent compound with low cytotoxicity that markedly reduced the synthesis of type I IFNs in response to STING pathway agonists. Besides, apigenin markedly suppressed innate immune responses triggered by the STING agonist SR-717. Mechanistically, apigenin downregulated IFN beta 1 (IFNB1) expression mediated by the STING pathway via dose-dependent inhibition of STING expression, reduction of dimerization, nuclear translocation of phosphorylated IRF3, and disruption of the association between STING and IRF3. Moreover, apigenin effectively mitigated pathological pulmonary inflammation and lung edema in lipopolysaccharide (LPS)-induced ALI in mice. Apigenin further strongly attenuated the hallmarks of immoderate inflammation (interleukin (IL)-6, IL-1[Formula: see text], and tumor necrosis factor [Formula: see text]) and innate immune responses (IFNB1, C-X-C motif chemokine ligand 10, and IFN-stimulated gene 15) by preventing the activation of the STING/IRF3 pathway both in vitro and in vivo. Importantly, SR-717 significantly reversed the inhibitory effects of apigenin in LPS-induced THP1-BlueTM ISG macrophages. Collectively, apigenin effectively alleviated innate immune responses and mitigated inflammation in LPS-induced ALI via inhibition of the STING/IRF3 pathway. These findings suggest the potential of apigenin as a prophylactic and therapeutic candidate for managing STING-mediated diseases.

BRD4-binding enhancer promotes CRC progression by interacting with YY1 to activate the Wnt pathway through upregulation of TCF7L2.

Colorectal carcinoma (CRC), one of the most life-threatening cancer types, is associated with aberrant expression of epigenetic modifiers and activation of the Wnt pathway. However, the role of epigenetic regulators in driving cancer cell proliferation and their potential as therapeutic targets affecting the Wnt pathway remain unclear. In this study, BRD4 was found to promote the progression of CRC both in vitro and in vivo. The expression of BRD4 correlated with shortened CRC patient survival. In addition, BRD4 function was strongly correlated with the Wnt pathway, but rather through regulation of TCF7L2 at transcriptional levels. BRD4 and H3K27ac have overlapping occupancies in the cis-regulatory elements of TCF7L2, suggesting enhancer-based epigenetic regulation. Numerous YY1 binding sites were found in the abovementioned region. YY1 recruited BRD4 to bind to cis-regulatory elements of TCF7L2, thereby regulating the expression of TCF7L2. Altogether, this study validates that BRD4 performs a canonical epigenetic regulatory function in CRC and can be used in the treatment of Wnt pathway-dependent CRC or other malignancies with clinically available bromodomain inhibitors.

SNHG3/hsa-miR-455-5p Axis-mediated High Expression of MTHFD2 Correlates with Tumor Immune Infiltration and Endometrial Carcinoma Progression.

Purpose: Endometrial carcinoma (EC) is one of the three most common female genital tract cancers, and it contributes to the leading deaths of gynecologic cancer. MTHFD2 was reported up-regulated and associated with poor prognosis in many malignancies. However, its biological functions and mechanisms in EC are unclear. The present study aimed to identify the biological functions and potential molecular mechanisms of MTHFD2 in EC. Methods: The gene expression and information of patients used in this study were derived from TCGA, GEO and HPA databases. KM survival analysis was used to explore the clinical outcomes of EC patients and correlation analysis was applied to find the correction between MTHFD2 expression level and immune infiltration in EC. We used GO and GSEA analysis to explore the biological functions and mechanisms of MTHFD2. The CCK8 assay, the colony formation assay and the transwell migration assay were conducted to validate the function of MTHFD2 in EC cells. We applied STRING to find the protein that interacted with MTHFD2. Finally, ENCORI was used to explore the potential upstream regulation of MTHFD2 in EC and it was validated in EC cells. Results: In the present study, we found that MTHFD2 was up-regulated in EC and its high expression level was associated with patients' poor prognosis and adverse clinical parameters. MTHFD2 level was shown to be correlated with immune infiltration. Knockdown of MTHFD2 inhibited the malignant phenotype of HEC-1A and Ishikawa cells, including proliferation, colony formation and migration. Furthermore, we found the SNHG3/hsa-miR-455-5p axis as the potential upstream of MTHFD2. Conclusion: SNHG3/hsa-miR-455-5p axis-mediated high expression of MTHFD2, and the MTHFD2 expression level was associated with tumor immune infiltration and endometrial carcinoma progression. Knockdown of MTHFD2 significantly inhibited the malignant phenotype of EC cells. MTHFD2 may be a valuable predictive biomarker, and targeting MTHFD2 may be an effective way to improve the therapeutic effect in EC.

Inverted U-shaped relationships between bone mineral density and VCTE-quantified degree of hepatic steatosis in adolescents: Evidence from the NHANES.

INTRODUCTION: There may be inaccuracies in hepatic steatosis in past research assessing the relationship between bone metabolism and liver steatosis. The goal of the current research was to look at the associations between bone mineral density (BMD) and the hepatic steatosis and fibrosis as detected by vibration-controlled transient elastography (VCTE) in teenagers in the United States. METHODS: Weighted multiple linear regression models and smoothed curve fitting were used to investigate the association between BMD and the degree of hepatic steatosis and fibrosis in adolescents. RESULTS: In 829 adolescents aged 12-19 years we found a negative association between total BMD and CAP (controlled attenuation parameter) [-32.46 (-58.98, -9.05)] and a significant positive association between lumbar BMD and LSM (liver stiffness measurement) [1.35 (0.19, 2.51)]. The inverted U-shaped relationships were founded between total BMD, lumbar BMD, pelvis BMD, and CAP with inflection points of 221.22 dB/m, 219.88 dB/m, and 216.02 dB/m, respectively. CONCLUSIONS: In adolescents, higher BMD is significantly associated with lower levels of hepatic steatosis and higher levels of liver stiffness.

Ideal entry point and direction of retrograde intramedullary nailing of the tibia.

PURPOSE: To determine the ideal entry point and direction of retrograde intramedullary nailing of the tibia. METHODS: The imaging data of patients with distal tibial fractures from June 2020 to December 2021 in our hospital were collected, and computer-aided design was performed. The relevant data were imported into the software for processing, so as to obtain a distal tibial fracture model and simulate the retrograde intramedullary nail placement in the tibia. The entry points and angles at which the intramedullary nail could be inserted successfully and the fracture could be maintained in good alignment were overlapped and counted to obtain the safe entry range and angle. The center of this safe range is the ideal entry point for retrograde intramedullary nailing of the tibia, and the mean value of the angle is the ideal direction of entry. RESULTS: The ideal entry point of the retrograde intramedullary nailing was located at the midpoint of the medial malleolus in the C-arm fluoroscopic anteroposterior (AP) and lateral view. The ideal nail entry direction was located at the anatomic axis of the medial malleolus in the AP position and at the anatomic axis of the distal tibial metaphysis in the lateral position. CONCLUSION: The ideal point and direction of nail insertion for retrograde tibial intramedullary nailing is a "double midpoint, double axis" approach.

Associations between weight-adjusted waist index and fractures: a population-based study.

INTRODUCTION: The weight-adjusted waist circumference index (WWI) is a novel obesity evaluation indicator that appears to be superior to body mass index (BMI) and waist circumference (WC) in evaluating muscle and fat mass. The purpose of this study was to investigate the association between WWI and fractures among adults. METHODS: In this cross-sectional study, multivariate logistic regression and smoothed curve fitting were used to investigate linear and nonlinear associations between WWI and fractures, based on data from 28,679 adult participants in the National Health and Nutrition Examination Survey (NHANES) from 1999 to 2018. RESULTS: After adjusting for all covariates, the prevalence of hip/wrist/spine fractures among all participants was 1.09%, 8.87%, and 1.97%, respectively. A 1-unit increase in WWI was associated with a 5% increase in the odds of hip fractures [1.05 (1.01, 1.10)], and a 9% increase in the odds of spine fractures [1.09 (1.06, 1.13)], but not with the prevalence of wrist fractures [0.97 (0.94, 1.06)]. CONCLUSIONS: Higher WWI was associated with an increased prevalence of hip fracture and spine fracture, but not wrist fracture.

BMI, socioeconomic status, and bone mineral density in U.S. adults: Mediation analysis in the NHANES.

Introduction: The mechanism by which socioeconomic status (SES) affects bone mineral density (BMD) remains unknown, and body mass index (BMI) may be a potential mediator. The purpose of this study was to investigate whether BMI mediates the relationship between SES [education level and poverty income ratio (PIR)] and lumbar BMD and the proportion it mediates. Methods: This study included a total of 11,075 adults from the National Health and Nutrition Examination Survey (NHANES). Lumbar BMD was measured at the lumbar spine by dual-energy X-ray absorptiometry (DXA). Multivariate linear regression and smoothing curve fitting were used to investigate the relationship between SES and lumbar BMD. Mediator analysis was used to investigate the proportion of BMI mediating the association between SES and BMD. Results: In the fully adjusted model, there was a positive correlation between SES and BMD (education level: ß = 0.025, 95% CI: 0.005, 0.045; PIR: ß = 0.007, 95% CI: 0.002, 0.011). Mediation analysis showed that BMI mediated the relationship between PIR, education level, and lumbar BMD with a range of mediation proportions from 13.33 to 18.20%. Conclusion: BMI partially mediated the positive association between SES and BMD, and this association may be largely mediated by factors other than BMI.

c-Jun phosphorylated by JNK is required for protecting Gli2 from proteasomal-ubiquitin degradation by PGE2-JNK signaling axis.

Hedgehog (Hh) signaling pathway includes canonical and non-canonical activation manners. In colorectal cancer, we have previously shown that PGE2-JNK could initiate non-canonical activation of the Hh signaling pathway. In this study, we showed that c-Jun, a classic substrate of JNK, increased Gli2 protein stability after phosphorylated by PGE2. Suppressing the function of c-Jun or JNK indicated that c-Jun prevents Gli2 from protease degradation caused by PGE2-JNK. Moreoer, we revealed that less ubiquitination of Gli2 was detected in colorectal cancer cells treated with PGE2 while suppression of c-Jun restored the ubiquitination of Gli2. In addition, we observed that suppression of c-Jun significantly decreased Gli2 expression no matter when Gli2 remained in phosphorylation or non-phosphorylation state. These phenomena were recapitulated, when the endpoint of Gli2 expression was replaced by Gli2 ubiquitination. Furthermore, we demonstrated that restricting c-Jun function ablated the PGE2-provoked Hh activity and proliferation of colorectal cancer cells. These results elucidated that the evasion of Gli2 with phosphorylation from proteasomal-ubiquitin degradation needed the cooperation of phosphorylated c-Jun by kinase JNK, which contributed to promoting Hh activation and the proliferation of colorectal cancer cells. This study provides a theoretical foundation to target PGE2 downstream for the prevention and treatment of colorectal cancer.

The PROTAC selectively degrading Bcl-xL represents a novel Hedgehog pathway inhibitor with capacity of combating resistance to Smoothened inhibitors while sparing bone growth.

Rationale: Primary and acquired resistance to Smoothened (Smo) inhibitors largely hampered their clinical efficacy. Given the important functions of hedgehog (Hh) pathway in bone formation and development, the permanent defects in bone growth caused by Smo inhibitors further restrict the use of Smo inhibitors for pediatric tumor patients. Anti-apoptotic Bcl-2 proteins regulate Hh activity by engaging a Bcl-2 homology (BH) domain sequence found in suppressor of fused (Sufu). In this study, we tested the effect of SIAIS361034, a Proteolysis Targeting Chimera (PROTAC) specifically targeting B-cell lymphoma extra large (Bcl-xL) to the celeblon (CRBN) E3 ligase for degradation, on combating the resistance and reducing the toxicity of bone growth caused by Hh inhibition. Methods: Fluorescence polarization, homogeneous time-resolved fluorescence (HTRF) assay, immunoblot, and immunoprecipitation (IP) were used to evaluate whether SIAIS361034 is an appropriate Bcl-xL PROTAC. Dual luciferase reporter assay, real-time quantitative PCR (RT-qPCR), depilatory model, and SmoA1 model were established to assess the effect of SIAIS361034 on the activity of Hh signaling pathway and its ability to overcome drug resistance in vitro and in vivo. Molecular mechanisms of SIAIS361034 for inhibiting Hh activity were demonstrated by dual luciferase reporter assay, immunoblot, and immunofluorescence staining. PET-CT and histopathology of bone tissues were used to assess the effects of SIAIS361034 on bone growth. Results: We observed that SIAIS361034 efficiently and selectively inhibits the activity of the Hh pathway in vitro and in vivo, by interrupting Bcl-xL/Sufu interaction, therefore, promoting the interaction of Sufu with Gli1. Moreover, SIAIS361034 possesses the ability of combating resistance to current Smo inhibitors caused by Smo mutations and Gli2 amplification and remarkably inhibits the growth of SmoA1 tumors in vivo. In contrast to von Hippel-Lindau (VHL) E3 ligase, our result further reveals little detectable expression of CRBN in two types of cells critical for bone development, human articular chondrocytes and human fetal osteoblastic cells. Moreover, treatment with SIAIS361034 results in no impairment on the bone growth of young mice, accompanying no alteration of the expression of Bcl-xL and Gli1 proteins. Conclusion: Our findings demonstrate that selectively targeting Bcl-xL by PROTAC is a promising strategy for combating resistance to Smo inhibitors without causing on-target drug toxicities of bone growth.

ABT-737 suppresses aberrant Hedgehog pathway and overcomes resistance to smoothened antagonists by blocking Gli.

Abnormally activated Hedgehog (Hh) pathway has been linked to multiple types of cancers including medulloblastoma (MB). Current Hh-targeted drug development projects mainly focus on antagonizing the upstream oncoprotein Smoothened (Smo). However, the effectiveness of Smo inhibitors is compromised by primary and acquired resistance, which is caused by mutations of Smo or other downstream components. Here, we conducted a cellular screening of small-molecule compounds and identified ABT-737 as a selective Hh inhibitor resulting in active suppression of human Hh-dependent MB cells. Mechanistically, ABT-737 suppressed Hh signals far-downstream of Smo and Sufu at Gli transcriptional effector level. In line with this, ABT-737 potentially inhibited wild-type and drug-resistant mutant Smo. More importantly, ABT-737 also delayed the growth of drug-refractory Hh-dependent MB xenografts derived from genetically engineered mouse model in vivo. These findings identify ABT-737 as a therapeutical substance for cancers with excessive Hh signaling activity, especially for those with primary or acquired resistance to Smo inhibitors in clinic.

Novel Retrograde Tibial Intramedullary Nailing for Distal Tibial Fractures.

Purpose: Postoperative distal tibial fractures are often associated with complications such as difficulties in fracture healing and surgical incision infection. The purpose of this study is to evaluate the fracture healing time and functional recovery after a retrograde tibial intramedullary nail treatment for distal tibial fractures. Methods: We retrospectively studied 9 cases of patients with distal tibial fractures treated with retrograde intramedullary nailing (IMN). Fracture healing time was based on monthly postoperative x-ray imaging results, and functional outcomes were defined according to the American Orthopaedic Foot and Ankle Society (AOFAS) ankle-hindfoot score. Results: Among the 9 patients with distal tibial fractures from July 2020 to April 2021, the mean age was 51.8 ± 13.8 years. The classification of distal tibial fractures in the 9 patients according to OTA (Orthopaedic Trauma Association) includes 6 extra-articular fractures (3 of type A1, 2 of type A2, and 1 of type A3) and 3 intra-articular fractures (1 of type C1 and 2 of type C2). Among them, there were 5 closed fracture cases and 4 open fracture cases (according to Gustilo classification: 2 of type I, 2 of type II). We treated the fractures surgically with new retrograde tibial intramedullary nailing. The mean follow-up time for this group was 7.9 months (5-12 months). According to monthly postoperative radiographs performed to monitor fracture healing, the mean healing time was 3.3 months (3-4 months). Final postoperative function according to the AOFAS ankle-hindfoot score shows 6 excellent cases, 3 good cases. No serious complications such as postoperative infection, bone and internal fixation exposure, osteofascial compartment syndrome, or vascular nerve injury occurred. Conclusion: The new retrograde tibial intramedullary nail (RTN) has some unique advantages, and its treatment of distal tibial fractures can achieve good efficacy, but further studies are still needed to verify it.

Saikosaponin B1 and Saikosaponin D inhibit tumor growth in medulloblastoma allograft mice via inhibiting the Hedgehog signaling pathway.

Medulloblastoma (MB), accounting for nearly 10% of all childhood brain tumors, are implicated with aberrant activation of the Hedgehog (Hh) signaling pathway. Saikosaponin B1 (SSB1) and Saikosaponin D (SSD), two bioactive constituents of Radix Bupleuri, are reported to have many biological activities including anticancer activities. In our work, we evaluated the inhibition of SSB1 and SSD on MB tumor growth in allograft mice and explored the underlying mechanisms. The associated biological activity was investigated in Shh Light II cells, an Hh-responsive fibroblast cell line, using the Dual-Glo® Luciferase Assay System. First, SSB1 (IC50, 241.8 nM) and SSD (IC50, 168.7 nM) inhibited GLI-luciferase activity in Shh Light II cells stimulated with ShhN CM, as well as Gli1 and Ptch1 mRNA expression. In addition, both compounds suppressed the Hh signaling activity provoked by smoothened agonist (SAG) or excessive Smoothened (SMO) expression. Meanwhile, SSB1 and SSD did not inhibit glioma-associated oncogene homolog (GLI) luciferase activity activated by abnormal expression of downstream molecules, suppressor of fuse (SUFU) knockdown or GLI2 overexpression. Consequently, SSB1 (30 mg/kg, ip) and SSD (10 mg/kg, ip) displayed excellent in vivo inhibitory activity in MB allografts, and the tumor growth inhibition ratios were approximately 50% and 70%, respectively. Our findings, thus, identify SSB1 and SSD significantly inhibit tumor growth in MB models by inhibiting the Hedgehog pathway through targeting SMO.

PGE2-JNK signaling axis non-canonically promotes Gli activation by protecting Gli2 from ubiquitin-proteasomal degradation.

Both bench and bedside investigations have challenged the supportive role of Hedgehog (Hh) activity in the progression of colorectal cancers, thus raising a critical need to further deeply determine the contribution of Hh to the growth of colorectal cancer. Combining multiple complementary means, including in vitro and in vivo inflammatory colorectal cancer models, and pathological analysis of clinical colorectal cancer patients samples. We report that colorectal cancer cells hijack prostaglandin E2 (PGE2) to non-canonically promote Hh transcriptional factor Gli activity and Gli-dependent proliferation of colorectal cancer cells in a Smo-independent manner. Mechanistically, PGE2 activates c-Jun N-terminal kinase (JNK), which in turn enables Gli2 to evade ubiquitin-proteasomal degradation by phosphorylating Gli2 at Thr1546. This study not only presents evidence for understanding the contribution of Hh to colorectal cancers, but also provides a novel molecular portrait underlying how PGE2-activated JNK fine-tunes the evasion of Gli2 from ubiquitin-proteasomal degradation. Therefore, it proposes a rationale for the future evaluation of chemopreventive and selective therapeutic strategies for colorectal cancers by targeting PGE2-JNK-Gli signaling route.

Circ_0016347 Promotes Osteosarcoma Progression by Regulating miR-1225-3p/KCNH1 Axis.

Background: Osteosarcoma (OS) is a common malignant bone cancer and usually occurs in adolescents and children. Circular RNAs (circRNAs) play essential roles in tumor development and progression. This study aimed to explore the function and molecular basis of circ_0016347 in OS progression. Materials and Methods: The levels of circ_0016347, miR-1225-3p, and ether à go-go 1 (KCNH1) were measured by quantitative real-time polymerase chain reaction or western blot assay. Cell proliferation was assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and colony formation assay. Cell migration and invasion were evaluated by transwell assay. Glucose consumption and lactate production were detected by glucose detection and lactic acid detection kits. The levels of Ki-67, matrix metalloproteinase-9 (MMP-9), and hexokinase-2 (HK2) were examined by western blot assay. The interaction among circ_0016347, miR-1225-3p, and KCNH1 was validated by dual-luciferase reporter assay. Xenograft assay was conducted to analyze tumor growth in vivo. Results: Circ_0016347 and KCNH1 were upregulated, while miR-1225-3p was downregulated in OS tissues or cells. Circ_0016347 and KCNH1 promoted proliferation, migration, invasion, and glycolysis of OS cells. Circ_0016347 regulated OS progression by modulating KCNH1. Circ_0016347 was a sponge of miR-1225-3p, and miR-1225-3p targeted KCNH1. Circ_0016347 regulated KCNH1 expression via sponging miR-1225-3p. Moreover, silencing of circ_0016347 inhibited tumor growth in vivo. Conclusion: Circ_0016347 contributed to OS progression through the miR-1225-3p/KCNH1 axis, which might provide a promising biomarker for OS therapy.

Development of hedgehog pathway inhibitors by epigenetically targeting GLI through BET bromodomain for the treatment of medulloblastoma.

Medulloblastoma (MB) is a common yet highly heterogeneous childhood malignant brain tumor, however, clinically effective molecular targeted therapy is lacking. Modulation of hedgehog (HH) signaling by epigenetically targeting the transcriptional factors GLI through bromodomain-containing protein 4 (BRD4) has recently spurred new interest as potential treatment of HH-driven MB. Through screening of current clinical BRD4 inhibitors for their inhibitory potency against glioma-associated oncogene homolog (GLI) protein, the BRD4 inhibitor 2 was selected as the lead for further structural optimization, which led to the identification of compounds 25 and 35 as the high potency HH inhibitors. Mechanism profiling showed that both compounds suppressed HH signaling by interacting with the transcriptional factor GLI, and were equally potent against the clinical resistant mutants and the wild type of smoothened (SMO) receptor with IC50 values around 1 nmol/L. In the resistant MB allograft mice, compound 25 was well tolerated and markedly suppressed tumor growth at both 5 mg/kg (TGI = 83.3%) and 10 mg/kg (TGI = 87.6%) doses. Although further modification is needed to improve the pharmacokinetic (PK) parameters, compound 25 represents an efficacious lead compound of GLI inhibitors, possessing optimal safety and tolerance to fight against HH-driven MB.

Berberine inhibits colorectal tumor growth by suppressing SHH secretion.

Hedgehog plays an important role in a wide range of physiological and pathological conditions. Paracrine activation of Hedgehog pathway in stromal cells increases the expression of VEGF, which promotes neovascularization in colorectal cancer and ultimately the growth of colorectal cancer. Berberine (BBR) has anticancer activity. In this study we investigated whether BBR inhibited the growth of colon cancer through suppressing the paracrine sonic hedgehog (SHH) signaling in vitro and in vivo. We showed that BBR (1-10 µM) dose-dependently inhibited the secretion and expression of SHH protein in HT-29 and SW480 cells. BBR did not influence the transcription of SHH, but promoted the degradation of SHH mRNA, thus decreased the SHH mRNA expression in the colorectal cancer cells. In nude mice bearing HT-29 xenograft, oral administration of BBR (100 mg · kg-1 · d-1) or a positive control drug GDC-0449 (100 mg · kg-1 · d-1) for 4 weeks markedly suppressed the growth of HT-29 tumor with BBR exhibiting a better antitumor efficacy. The tumor growth inhibition caused by BBR or GDC-0449 was comparable to their respective inhibitory effect on the mouse-specific Gli mRNA expression in the tumor. However, BBR (20 µM) did not affect the expression of human transcription factor Gli1 mRNA in HT-29 and SW480 cells. In conclusion, BBR promotes the degradation of SHH mRNA in colorectal cancer cells, interrupting the paracrine Hedgehog signaling pathway activity thus suppresses the colorectal cancer growth. This study reveals a novel molecular mechanism underlying the anticancer action of BBR.

ABT-199 inhibits Hedgehog pathway by acting as a competitive inhibitor of oxysterol, rather as a BH3 mimetic.

Aberrantly activated Hedgehog (Hh) pathway is critical for driving the initiation and progression of multiple types of cancers, including medulloblastoma (MB) and basal cellular carcinoma (BCC). The majority of current Hh antagonist function by targeting the transmembrane domain of the oncoprotein Smoothened (Smo), a G-protein-coupled receptor-like receptor of Hh pathway. However, the primary and acquired resistance to current Smo inhibitors raise a critical need to develop next-generation of Smo inhibitors to improve their clinical efficacy. In this study, we identify that FDA approved drug ABT-199 significantly and selectively inhibits the Hh pathway. Mechanistically, ABT-199 acts as a competitive inhibitor of oxysterol by potentially targeting the cysteine rich domain (CRD) of Smo, rather as a BH3 mimetic. ABT-199 obviously inhibits the growth of Hh-driven tumors and possesses capacity of combating the primary and acquired resistance to Smo inhibitors caused by Smo mutations. Our data reposition ABT-199 as a Smo inhibitor for treating Hh-driven tumors, especially for those bearing Smo mutations and resistant to current Smo inhibitors. Meanwhile, our findings strengthen the argument that the CRD of Smo is a promising target for developing novel Smo inhibitors with capacity of combating the resistance to Smo inhibitors.

Discovery of Small Molecule Inhibitors Targeting the Sonic Hedgehog.

The aberrant activation of hedgehog (Hh) signaling pathway is closely related to human diseases. The upstream protein, N-terminal product of sonic hedgehog (ShhN) is overexpressed in many cancers and considered as a promising antitumor target. Inhibitors that bind to ShhN and break its interaction with the 12-transmembrane glycoprotein patched (Ptch) protein are highly wanted to tune down the abnormal Hh pathway activation. However, research of ShhN inhibitors remains lacking. In this paper, we computationally screened potential inhibitors against the ShhN-Ptch interaction interface, and tested their activities by experimental studies. Seven compounds (1-7) with diverse scaffolds, showed inhibition in cellular assays and directly bound to ShhN in vitro. The compounds were verified to modulate the Hh pathway activity. Furthermore, we studied the structure-activity relationship of the pyrimidine (7) derivatives and identified a potent compound (7_3d3) with IC50 of 0.4 ± 0.1 µM in cellular assays. These small molecule inhibitors of ShhN provide novel chemical probes for future investigations of Hh signaling.

Expression of HIF-1α is a predictive marker of the efficacy of neoadjuvant chemotherapy for locally advanced cervical cancer.

Platinum-based, arterial infusion chemotherapy as a neoadjuvant chemotherapy (NACT) followed by hysterectomy may be efficient for the treatment of locally advanced cervical cancer and improve prognosis. It is important to predict whether the NACT would be effective before it is launched. Hypoxia inducible factor-1α (HIF-1α) is the master transcriptional regulator of the cellular response to altered oxygen concentration. HIF-1α protein expression is elevated in numerous human malignancies, contributes to poor disease outcome, and has been reported to induce tumorigenesis and chemoresistance. In the present study, patients with International Federation of Gynecology and Obstetrics stage IIB-IIIB cervical cancer (n=59) between 2008 and 2014 were assessed for HIF-1α expression by immunohistochemistry. Tumor samples were obtained by biopsy before any treatment. A double-path chemotherapy regimen, paclitaxel (intravenous) plus cisplatin (intra-arterial injection into the uterine region), was used as NACT. The patients were then separated into two groups according to NACT response: One group comprised patients with NACT, for whom the response to treatment was efficient resulting in complete/partial remission of the tumor (CR + PR group; n=52), the other group contained patients with NACT, for whom the result of the treatment was a stable/progressive disease (SD + PD group; n=7). HIF-1α expression was tested in paraffin-embedded sections using immunohistochemistry. HIF-1α expression was significantly higher in the SD + PD group compared with the CR + PR group (P=0.029). The overall survival time was significantly longer in the CR + PR group compared with the SD + PD group (P<0.001). When the patients were divided into two groups based on HIF-1α expression levels. Low (weighted score ≤4, n=39) and high (weighted score ≥6, n=20) expression level groups; the low HIF-1α expression group was significantly more susceptible to NACT treatment (P=0.025). Cox hazard analysis revealed that a high level of HIF-1α expression and lymph node metastases were significant independent predictors of poor overall survival (P=0.025, HR=6.354; P=0.020, HR=6.909, respectively). These results indicated that the expression of HIF-1α may be able to predict the efficiency of NACT and may be considered an independent prognostic factor for stage IIB-IIIB cervical cancer.