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Chemokine (C-C motif) ligand 2 (CCL2) has been shown to play an important role in the regulation of tumor cell growth, metastasis and host immune response. CCL2 preferentially binds to the C-C chemokine receptor type 2 (CCR2), which is expressed in various tissues. However, the role of the CCL2/CCR2 axis in hepatocellular carcinoma (HCC) invasion and its molecular mechanisms remain unclear. The aim of this study was to elucidate this issue. The human HCC cell line MHCC-97H was treated with CCL2. Cyclopamine, a smoothened (SMO) antagonist, was used to inhibit SMO activity. CCR2 siRNA and Gli-1 siRNA were used to inhibit CCR2 and Gli-1 expression respectively. The effect of CCL2 and Hedgehog (Hh) signaling on cancer cell epithelial-mesenchymal transition (EMT) and invasion was evaluated by quantitative real-time PCR analysis, western blotting and Transwell invasion assay. Our results revealed that CCL2 induced HCC cell invasion and EMT. This effect was accompanied by the activation of Hh signaling, the upregulation of Snail and vimentin and the reduction of E-cadherin. Notably, prior silencing of CCR2 with siRNA abolished CCL2-induced Hh signaling activation, Snail and vimentin upregulation, E-cadherin reduction, as well as HCC cell invasion and EMT. Furthermore, pretreatment with cyclopamine or predepletion of Gli-1 by siRNA also eliminated the changes of Snail, vimentin and E-cadherin, and HCC invasion and EMT caused by CCL2. Collectively, our results revealed that the link between the CCL2/CCR2 axis and the Hh pathway plays an important role in HCC progression. Therefore, the CCL2/CCR2 axis may represent a promising therapeutic target to prevent HCC progression.

The autonomic nervous system contributes to prostate cancer proliferation and metastasis. However, the exact molecular mechanism remains unclear. In this study, muscarinic acetylcholine receptor M1 (CHRM1) expression was measured via immunohistochemical analysis in human prostate cancer tissue array slides. PC-3, LNCaP, and A549 cells were treated with pirenzepine or carbachol, and the cell migration and invasion abilities were evaluated. Western blotting and quantitative real-time PCR were performed to measure GLI family zinc finger 1 (GLI1), patched 1 (PTCH1), and sonic hedgehog (SHH) expression levels. High expression of CHRM1 was found in early-stage human prostate cancer tissues. In addition, the selective CHRM1 antagonist pirenzepine inhibited PC-3, LNCaP, and A549 cell migration and invasion, but the agonist carbachol promoted the migration and invasion of these three cell lines. Muscarinic signaling can be relayed by hedgehog signaling. These data show that CHRM1 is involved in the regulation of prostate cancer migration and invasion through the hedgehog signaling pathway.

Due to its importance in the pathogenesis of oral squamous cell carcinoma (OSCC), the Hedgehog (HH) pathway is considered a potential therapeutic target. We investigated the effects of GANT61, a GLI inhibitor, on HH gene expression, as well as on metastatic OSCC cell proliferation and death. Following culture in DMEM medium, cytotoxicity of GANT61 against different tumor and non-tumor cell types was assessed by alamarBlue assays. Cytotoxicity analysis revealed that the metastatic HSC3 cell line was the most sensitive (IC50: 36 µM) to the tested compound. The compound’s effects on the expression of HH pathways components were analyzed by qPCR and Western blot; cell viability was analyzed by trypan blue assay and flow cytometry were used to investigate cell cycle phase, morphology, and death patterns in HSC3 cells. A significant reduction in mRNA levels of the GLI1 transcription factor was found after 12 h of treatment withGANT61. Protein expression levels of other HH pathway components (PTCH1, SHH, and Gli1) and HSC3 cell viability also decreased after 24 h of treatment. Cell cycle analysis and death pattern evaluations revealed significantly increased nuclear fragmentation in sub-G1 phase, as well as cell death due to apoptosis. In conclusion, the significantly reduced GLI1 gene expression seen in response to the GLI inhibitor indicates diminished downstream activation in HH pathway components. GANT61 significantly reduced cell viability in the metastatic cell line of OSCC and promoted a significant increase in nuclear fragmentation and cell death by apoptosis.

Cilium, an organelle with a unique proteome and organization, protruding from the cell surface, generally serves as a force generator and signaling compartment. During ciliogenesis, ciliary proteins are synthesized in cytoplasm and transported into cilia by intraflagellar transport (IFT) particles, where the inner counterparts undergo reverse trafficking. The homeostasis of IFT plays a key role in cilial structure assembly and signaling transduction. Much progress has been made on the mechanisms and functions of IFT; however, recent studies have revealed the involvement of IFT particle subunits in organogenesis and spermatogenesis. In this review, we discuss new concepts concerning the molecular functions of IFT protein IFT25 and how its interactions with other IFT particle subunits are involved in mammalian development and fertility.

Many cellular processes are regulated by proteasome-mediated protein degradation, including regulation of signaling pathways and gene expression. Among the pathways regulated by the ubiquitin–proteasome system is the Hedgehog pathway and its downstream effectors, the Gli transcription factors. Here we provide evidence that proteasomal activity is necessary for maintaining the activation of the Hedgehog pathway, and this crucial event takes place at the level of Gli proteins. We undertook extensive work to demonstrate the specificity of the observed phenomenon by ruling out the involvement of primary cilium, impaired nuclear import, failed dissociation from Sufu, microtubule stabilization, and stabilization of Gli repressor forms. Moreover, we showed that proteasomal-inhibition-mediated Hedgehog pathway downregulation is not restricted to the NIH-3T3 cell line. We demonstrated, using CRISPR/Ca9 mutagenesis, that neither Gli1, Gli2, nor Gli3 are solely responsible for the Hedgehog pathway downregulation upon proteasome inhibitor treatment, and that Cul3 KO renders the same phenotype. Finally, we report two novel E3 ubiquitin ligases, Btbd9 and Kctd3, known Cul3 interactors, as positive Hedgehog pathway regulators. Our data pave the way for a better understanding of the regulation of gene expression and the Hedgehog signaling pathway.

Bone marrow-derived mesenchymal stem cells differentiate into neurons under the induction of Schwann cells. However, key microRNAs and related pathways for differentiation remain unclear. This study screened and identified differentially expressed microRNAs in bone marrow-derived mesenchymal stem cells induced by Schwann cell-conditioned medium, and explored targets and related pathways involved in their differentiation into neuronal-like cells. Primary bone marrow-derived mesenchymal stem cells were isolated from femoral and tibial bones, while primary Schwann cells were isolated from bilateral saphenous nerves. Bone marrow-derived mesenchymal stem cells were cultured in unconditioned (control group) and Schwann cell-conditioned medium (bone marrow-derived mesenchymal stem cell + Schwann cell group). Neuronal differentiation of bone marrow-derived mesenchymal stem cells induced by Schwann cell-conditioned medium was observed by time-lapse imaging. Upon induction, the morphology of bone marrow-derived mesenchymal stem cells changed into a neural shape with neurites. Results of quantitative reverse transcription-polymerase chain reaction revealed that nestin mRNA expression was upregulated from 1 to 3 days and downregulated from 3 to 7 days in the bone marrow-derived mesenchymal stem cell + Schwann cell group. Compared with the control group, microtubule-associated protein 2 mRNA expression gradually increased from 1 to 7 days in the bone marrow-derived mesenchymal stem cell + Schwann cell group. After 7 days of induction, microRNA analysis identified 83 significantly differentially expressed microRNAs between the two groups. Gene Ontology analysis indicated enrichment of microRNA target genes for neuronal projection development, regulation of axonogenesis, and positive regulation of cell proliferation. Kyoto Encyclopedia of Genes and Genomes pathway analysis demonstrated that Hippo, Wnt, transforming growth factor-beta, and Hedgehog signaling pathways were potentially associated with neural differentiation of bone marrow-derived mesenchymal stem cells. This study, which carried out successful microRNA analysis of neuronal-like cells differentiated from bone marrow-derived mesenchymal stem cells by Schwann cell induction, revealed key microRNAs and pathways involved in neural differentiation of bone marrow-derived mesenchymal stem cells. All protocols were approved by the Animal Ethics Committee of Institute of Radiation Medicine, Chinese Academy of Medical Sciences on March 12, 2017 (approval number: DWLI-20170311).

Somatic cell reprogramming and tissue repair share relevant factors and molecular programs. Here, Dickkopf‐3 (DKK3) is identified as novel factor for organ regeneration using combined transcription‐factor‐induced reprogramming and RNA‐interference techniques. Loss of Dkk3 enhances the generation of induced pluripotent stem cells but does not affect de novo derivation of embryonic stem cells, three‐germ‐layer differentiation or colony formation capacity of liver and pancreatic organoids. However, DKK3 expression levels in wildtype animals and serum levels in human patients are elevated upon injury. Accordingly, Dkk3‐null mice display less liver damage upon acute and chronic failure mediated by increased proliferation in hepatocytes and LGR5+ liver progenitor cell population, respectively. Similarly, recovery from experimental pancreatitis is accelerated. Regeneration onset occurs in the acinar compartment accompanied by virtually abolished canonical‐Wnt‐signaling in Dkk3‐null animals. This results in reduced expression of the Hedgehog repressor Gli3 and increased Hedgehog‐signaling activity upon Dkk3 loss. Collectively, these data reveal Dkk3 as a key regulator of organ regeneration via a direct, previously unacknowledged link between DKK3, canonical‐Wnt‐, and Hedgehog‐signaling. Re‐activation of embryonic pathways is a hallmark of tissue repair after injury. During somatic reprogramming similar molecular patterns are re‐activated, thus providing an excellent platform to identify relevant factors in tissue repair. Here, Dickkopf‐3 (DKK3) is identified as key player of gastrointestinal tissue regeneration and repair via a DKK3‐mediated interplay of canonical Wnt‐ and Hedgehog‐signaling.

Background: The hedgehog signaling system (HHS) plays an important role in the regulation of cell proliferation and differentiation during the embryonic phases. However, little is known about the involvement of HHS in the malignant transformation of cells. This study aimed to detect the role of HHS in the malignant transformation of human bronchial epithelial (16HBE) cells. Methods: In this study, two microfluidic chips were designed to investigate cigarette smoke extract (CSE)-induced malignant transformation of cells. Chip A contained a concentration gradient generator, while chip B had four cell chambers with a central channel. The 16HBE cells cultured in chip A were used to determine the optimal concentration of CSE for inducing malignant transformation. The 16HBE cells in chip B were cultured with 12.25% CSE (Group A), 12.25% CSE + 5 μmol/L cyclopamine (Group B), or normal complete medium as control for 8 months (Group C), to establish the in vitro lung inflammatory-cancer transformation model. The transformed cells were inoculated into 20 nude mice as cells alone (Group 1) or cells with cyclopamine (Group 2) for tumorigenesis testing. Expression of HHS proteins was detected by Western blot. Data were expressed as mean ± standard deviation. The t-test was used for paired samples, and the difference among groups was analyzed using a one-way analysis of variance. Results: The optimal concentration of CSE was 12.25%. Expression of HHS proteins increased during the process of malignant transformation (Group B vs. Group A, F = 7.65, P < 0.05). After CSE exposure for 8 months, there were significant changes in cellular morphology, which allowed the transformed cells to grow into tumors in 40 days after being inoculated into nude mice. Cyclopamine could effectively depress the expression of HHS proteins (Group C vs. Group B, F = 6.47, P < 0.05) and prevent tumor growth in nude mice (Group 2 vs. Group 1, t = 31.59, P < 0.01). Conclusions: The activity of HHS is upregulated during the CSE-induced malignant transformation of 16HBE cells. Cyclopamine can effectively depress expression of HHS proteins in vitro and prevent tumor growth of the transformed cells in vivo.

Aim of the study: Examination of copy number changes in a group of intracranial germ cell tumors (GCTs) with particular focus on putative aberrations of the main genes coding SHh pathway proteins. Material and methods: The study was performed on DNA isolated from fresh-frozen tumor tissue samples from eight GCTs, including six intracranial GCTs. The intracranial group consisted of three germinomas, two mature teratomas and one mixed germ cell tumor. Comparative genomic profiling analysis was carried out using microarray-CGH method (Cytosure ISCA UPD 4×180k, OGT). The results were analyzed with Feature Extraction (Agilent Technologies) and Nexus Copy Number (BioDiscovery) softwares. Results and conclusions: Chromosomal aberrations were found in two intracranial germinomas. These tumors were characterized by complex genomic profiles encompassing chromosomes 7, 8, 9, 10, 11, 12, 16, 17 and 19. Common findings were gain at 12p13.33p11.1 of 35 Mbp and gain at 17q11.1q25.3 of 55 Mbp. In one tumor, also SHh (7q36.3), SMO (7q32.1) and GLI3 (7p14.1) copy gains occurred together with 9q21.11q34.3 loss, including PTCH1, all being elements of SHh signaling pathway. Moreover, both tumors showed various copy gain of genes being ligands, regulators, receptors or target genes of SHh (MTSS1; PRKACA and FKBP8) as well as gain of genes of SHh coopting WNT pathway (WNT3, WNT5B, WNT9B in both tumors; WNT16, WNT2 in pineal lesion). Further studies on larger group are needed to characterize SHh-related gene alterations in intracranial GCTs and for searching genotype-phenotype relations.

Hedgehog (Hh) signaling is a highly conserved pathway that plays a vital role during embryonic development. Recently, uncontrolled activation of this pathway has been demonstrated in various types of cancer. Therefore, Hh pathway inhibitors have emerged as an important class of anti-cancer agents. Unfortunately, however, their reputation has been tarnished by the emergence of resistance during therapy, necessitating clarification of mechanisms underlying the drug resistance. In this review, we briefly overview canonical and non-canonical Hh pathways and their inhibitors as targeted cancer therapy. In addition, we summarize the mechanisms of resistance to Smoothened (SMO) inhibitors, including point mutations of the drug binding pocket or downstream molecules of SMO, and non-canonical mechanisms to reinforce Hh pathway output. A distinct mechanism involving loss of primary cilia is also described to maintain GLI activity in resistant tumors. Finally, we address the main strategies to circumvent the drug resistance. These strategies include the development of novel and potent inhibitors targeting different components of the canonical Hh pathway or signaling molecules of the non-canonical pathway. Further studies are necessary to avoid emerging resistance to Hh inhibitors and establish an optimal customized regimen with improved therapeutic efficacy to treat various types of cancer, including basal cell carcinoma.