Homeodomain Transcription Factors Nkx2.2 and Pax6 as Novel Biomarkers for Meningioma Tumor Treatment.

Meningioma is a common brain tumour which has neither a specific detection nor treatment method. The Sonic hedgehog (Shh) cell signaling pathway is a crucial regulatory pathway of mammalian organogenesis and tumorigenesis including meningioma. Shh cell signalling pathway cascade function by main transcription factor Gli1 and which further regulates in its downstream to Pax6 and Nkx2.2. This current study is aimed to explore the regulation of the Sonic hedgehog-Gli1 cell signaling pathway and its potential downstream targets in meningioma samples. A total of 24 surgically resected meningioma samples were used in this current study.Cytological changes were assessed using electron microscopic techniques as well as hematoxylin & eosin and DAPI staining. The expression pattern of Gli1, Nkx2.2 and Pax6 transcription factors were determined by using immunohistochemistry. The mRNA expression was assessed using RT-qPCR assays. Later, the whole transcriptome analysis of samples was performed with the amploseq technique. Results were compared with those obtained in normal human brain tissue (or normal meninges). Compared to the normal human brain tissue, meningioma samples showed crowded nuclei with morphological changes. Transcription factor Nkx2.2 expressed highly in all samples (24/24, 100%). Twenty-one of the 24 meningiomas (88%) showed high Gli1 and Pax6 expression. Whole transcriptome analysis of two meningioma samples also exhibited a very high increase in Gli1 expression signal in meningioma samples as compare to normal control. Hence, we may conclude that the Shh-Gli1 pathway is aberrantly activated in meningioma cells and is canonically upregulating the expression of transcription factors Pax6 and Nkx2.2. Supplementary Information: The online version contains supplementary material available at 10.1007/s12291-022-01085-1.

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

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

Cadmium-induced neurodegeneration and activation of noncanonical sonic hedgehog pathway in rat cerebellum.

BACKGROUND: Cadmium is a nonessential toxic heavy metal, which enters the body easily and damages the cellular system. The sonic hedgehog (Shh) signaling pathway is one of the key regulatory pathways, which define neural growth and development. OBJECTIVES: This study aimed to explore how cadmium exposure affects neural activities, Shh signaling cascade, and its downstream target genes. METHODS: Total 18 male Wistar rats were randomly divided into two groups, control and test groups. Test rats were administered with 3 mg cadmium/kg body weight, while the control rats were treated with vehicle continuously for 28 days. Thereafter, rats were killed and the isolated brain samples were examined using oxidative stress assessment, histological and immunohistological behavioral assessment, polymerase chain reaction (PCR), and the comet assay. RESULTS: A disturbed oxidative balance, DNA damage, and an upregulated Shh signaling pathway were observed in cadmium-treated samples. Loss of structural integrity in cerebellum and loss of motor activity were observed in cadmium-treated rats.

Sonic hedgehog, Wnt, and brain-derived neurotrophic factor cell signaling pathway crosstalk: potential therapy for depression.

There are various theories to explain the pathophysiology of depression and support its diagnosis and treatment. The roles of monoamines, brain-derived neurotrophic factor (BDNF), and Wnt signaling are well researched, but sonic hedgehog (Shh) signaling and its downstream transcription factor Gli1 are not well studied in depression. Shh signaling plays a fundamental role in embryonic development and adult hippocampal neurogenesis and also involved in the growth of cancer. In this article, we summarize the evidence for the Shh signaling pathway in depression and the potential crosstalk of Shh with Wnt and BDNF. Antidepressants are known to upregulate the adult hippocampal neurogenesis to treat depression. Shh plays an important role in adult hippocampal neurogenesis, and its downstream signaling components regulate the synthesis of Wnt proteins. Moreover, the expression of Gli1 and Smo is downregulated in depression. BDNF and Wnt signaling are also regulated by various available antidepressants, so there is the possibility that Shh may be involved in the pathophysiology of depression. Therefore, the crosstalk between the Shh, Wnt, and BDNF signaling pathways is being discussed to identify the potential targets. Specifically, the potential role of the Shh signaling pathway in depression is explored as a new target for better therapies for depression.

Laminin-adherent versus suspension-non-adherent cell culture conditions for the isolation of cancer stem cells in the DAOY medulloblastoma cell line.

Medulloblastoma (MB) is a highly malignant tumor of childhood. MB seems to be initiated and maintained by a small group of cells, known as cancer stem cells (CSCs). The CSC hypothesis suggests that a subset of tumor cells is able to proliferate, sustain the tumor, and develop chemoresistance, all of which make of CSC an interesting target for new anticancer therapies. The MB cell line DAOY was cultured in suspension by a medullosphere traditional culturing method and in adherent conditions by laminin-pre-coated flasks and serum-free medium enriched with specific growth factors. An increase in the stem features was shown when cells were successively cultured in hypoxia conditions. By contrast, a reduction in these properties was appreciated when cells were exposed to differentiation conditions. In addition, the CD133+ and CD133- subpopulations were isolated from cells grown in laminin-pre-coated flasks, and in vitro experiments showed that the CD133+ fraction represented the stem population and it could have CSC with a higher probability than the CD133- fraction. We can conclude that the laminin culture method in adherent conditions and the medullosphere traditional culturing method in suspension are similarly good for obtaining stem-like cells in the DAOY cell line.

Epigenetic regulation of human hedgehog interacting protein in glioma cell lines and primary tumor samples.

Glioma constitutes one of the most common groups of brain tumors, and its prognosis is influenced by different genetic and epigenetic modulations. In this study, we demonstrated low or no expression of hedgehog interacting protein (HHIP) in most of the cell lines and primary glioma tumor samples. We further proceeded to promoter methylation study of this gene in the same cell lines and primary tumor samples and found 87 % (7/8) HHIP methylation in glioblastoma cell lines and 75 % (33/44) in primary tumor samples. These methylation pattern correlates with low or unexpressed HHIP in both cell lines and primary tumor samples. Our results suggest the possibility of epigenetic regulation of this gene in glioma, similarly to medulloblastoma, gastric, hepatic, and pancreatic cancers. Also, HHIP might be a diagnostic or prognostic marker in glioma and help to the detection of these tumors in early stages of disease.

Mesenchymal-Stem-Cell-Based Therapy against Gliomas.

Glioblastoma is the most aggressive, malignant, and lethal brain tumor of the central nervous system. Its poor prognosis lies in its inefficient response to currently available treatments that consist of surgical resection, radiotherapy, and chemotherapy. Recently, the use of mesenchymal stem cells (MSCs) as a possible kind of cell therapy against glioblastoma is gaining great interest due to their immunomodulatory properties, tumor tropism, and differentiation into other cell types. However, MSCs seem to present both antitumor and pro-tumor properties depending on the tissue from which they come. In this work, the possibility of using MSCs to deliver therapeutic genes, oncolytic viruses, and miRNA is presented, as well as strategies that can improve their therapeutic efficacy against glioblastoma, such as CAR-T cells, nanoparticles, and exosomes.

A Comprehensive Review of miRNAs and Their Epigenetic Effects in Glioblastoma.

Glioblastoma is the most aggressive form of brain tumor originating from glial cells with a maximum life expectancy of 14.6 months. Despite the establishment of multiple promising therapies, the clinical outcome of glioblastoma patients is abysmal. Drug resistance has been identified as a major factor contributing to the failure of current multimodal therapy. Epigenetic modification, especially DNA methylation has been identified as a major regulatory mechanism behind glioblastoma progression. In addition, miRNAs, a class of non-coding RNA, have been found to play a role in the regulation as well as in the diagnosis of glioblastoma. The relationship between epigenetics, drug resistance, and glioblastoma progression has been clearly demonstrated. MGMT hypermethylation, leading to a lack of MGMT expression, is associated with a cytotoxic effect of TMZ in GBM, while resistance to TMZ frequently appears in MGMT non-methylated GBM. In this review, we will elaborate on known miRNAs linked to glioblastoma; their distinctive oncogenic or tumor suppressor roles; and how epigenetic modification of miRNAs, particularly via methylation, leads to their upregulation or downregulation in glioblastoma. Moreover, we will try to identify those miRNAs that might be potential regulators of MGMT expression and their role as predictors of tumor response to temozolomide treatment. Although we do not impact clinical data and survival, we open possible experimental approaches to treat GBM, although they should be further validated with clinically oriented studies.

Role of Circular RNA in Brain Tumor Development.

Central nervous system tumors are a leading cause of cancer-related death in children and adults, with medulloblastoma (MB) and glioblastoma (GBM) being the most prevalent malignant brain tumors, respectively. Despite tremendous breakthroughs in neurosurgery, radiation, and chemotherapeutic techniques, cell heterogeneity and various genetic mutations impacting cell cycle control, cell proliferation, apoptosis, and cell invasion result in unwanted resistance to treatment approaches, with a 5-year survival rate of 70-80% for medulloblastoma, and the median survival time for patients with glioblastoma is only 15 months. Developing new medicines and utilizing combination medications may be viewed as excellent techniques for battling MB and GBM. Circular RNAs (circRNAs) can affect cancer-developing processes such as cell proliferation, cell apoptosis, invasion, and chemoresistance in this regard. As a result, several compounds have been introduced as prospective therapeutic targets in the fight against MB and GBM. The current study aims to elucidate the fundamental molecular and cellular mechanisms underlying the pathogenesis of GBM in conjunction with circRNAs. Several mechanisms were examined in detail, including PI3K/Akt/mTOR signaling, Wnt/-catenin signaling, angiogenic processes, and metastatic pathways, in order to provide a comprehensive knowledge of the involvement of circRNAs in the pathophysiology of MB and GBM.

Expression and epigenetic modulation of sonic hedgehog-GLI1 pathway genes in neuroblastoma cell lines and tumors.

It is well known that sonic hedgehog signaling pathway plays a vital role during early embryonic development. It is also responsible for stem cell renewal and development of several cancers like colorectal and breast carcinoma and major brain tumors as medulloblastoma and glioblastoma. The role of sonic hedgehog signaling in the development of neuroblastoma has not been thoroughly investigated. In this study, we attempted to determine the expression of Bmi-1 stem cell marker and of Shh pathway downstream target genes glioma-associated oncogene homolog 1 (GLI1), protein patched homolog 1 (PTCH1), Cyclin D2, plakoglobin (γ catenin), NK2 homeobox 2 (NKX2.2), paired box gene 6 (PAX6), secreted frizzled-related protein 1 (SFRP1), and hedgehog interacting protein (HHIP) in 11 neuroblastoma cell lines and 41 neuroblastoma samples. Also, inhibition of the pathway was performed genetically by GLI1 knockdown siRNA or chemically by cyclopamine. After inhibition, low transcript expression was detected in downstream target genes like PTCH1, in the cell lines. We further preformed promoter methylation studies of Cyclin D2, PTCH1, HHIP, and SFRP1 genes by melting curve analysis-based methylation assay (MCA-Meth) and methylation-specific PCR (MSP). Results revealed no methylation in Cyclin D2 gene promoter in neuroblastoma samples or in cell lines; one cell line (MHH-NB-11) showed PTCH1 methylation; 3/11 (27%) cell lines and 9/41 (22%) neuroblastoma samples showed HHIP methylation; and 3/11 (27%) cell lines and 11/41 (27%) samples showed SFRP1 methylation. Taken together, our results suggest the possibility of two levels of control of the sonic hedgehog signaling pathway: transcriptional and epigenetic, which might offer new therapeutic possibilities to modulate the pathway and try to suppress tumor growth.

Human hedgehog interacting protein expression and promoter methylation in medulloblastoma cell lines and primary tumor samples.

Medulloblastoma is the most common pediatric brain tumor and its development is affected by genetic and epigenetic factors. In this study we found there is low or no expression of the hedgehog interacting protein (HHIP), a negative regulator of the sonic hedgehog pathway, in most medulloblastoma cell lines and primary samples explored. We proceeded to promoter methylation assays of this gene by MCA-Meth, and found that HHIP was hypermethylated in all medulloblastoma cell lines, but only in 2 out of 14 (14%) primary tumor samples. Methylation correlated with low or unexpressed HHIP in cell lines but not in primary tumor samples. These results suggest the possibility of epigenetic regulation of HHIP in medulloblastoma, similarly to gastric, hepatic and pancreatic cancer. However, HHIP seems to be not only under regulation of promoter methylation, but under other factors involved in the control of its low levels of expression in medulloblastoma.

Glioblastoma and MiRNAs.

Glioblastoma (GB) is one of the most common types of lethal brain tumors. Although several treatment options are available including surgery, along with adjuvant chemo and radiotherapy, the disease has a poor prognosis and patients generally die within 14 months of diagnosis. GB is chemo and radio resistant. Thus, there is a critical need for new insights into GB treatment to increase the chance of therapeutic success. This is why microRNA (miRNA) is being potentially considered in the diagnosis and treatment of glioblastoma. The objective of our review is to provide a holistic picture of GB up-regulated and down-regulated miRNA, in relationship with the expression of other genes, cell signaling pathways, and their role in GB diagnosis and treatment. MiRNA treatment is being considered to be used against GB together with radiotherapy and chemotherapy. Moreover, the use of miRNA as a diagnostic tool has also begun. Knowing that miRNAs are isolated in almost all human body fluids and that there are more than 3000 miRNAs in the human genome, plus the fact that each miRNA controls hundreds of different mRNAs, there is still much study needed to explore how miRNAs relate to GB for its proliferation, progression, and inhibition.

Regulation of sonic hedgehog-GLI1 downstream target genes PTCH1, Cyclin D2, Plakoglobin, PAX6 and NKX2.2 and their epigenetic status in medulloblastoma and astrocytoma.

BACKGROUND: The Sonic hedgehog (Shh) signaling pathway is critical for cell growth and differentiation. Impairment of this pathway can result in both birth defects and cancer. Despite its importance in cancer development, the Shh pathway has not been thoroughly investigated in tumorigenesis of brain tumors. In this study, we sought to understand the regulatory roles of GLI1, the immediate downstream activator of the Shh signaling pathway on its downstream target genes PTCH1, Cyclin D2, Plakoglobin, NKX2.2 and PAX6 in medulloblastoma and astrocytic tumors. METHODS: We silenced GLI1 expression in medulloblastoma and astrocytic cell lines by transfection of siRNA against GLI1. Subsequently, we performed RT-PCR and quantitative real time RT-PCR (qRT-PCR) to assay the expression of downstream target genes PTCH1, Cyclin D2, Plakoglobin, NKX2.2 and PAX6. We also attempted to correlate the pattern of expression of GLI1 and its regulated genes in 14 cell lines and 41 primary medulloblastoma and astrocytoma tumor samples. We also assessed the methylation status of the Cyclin D2 and PTCH1 promoters in these 14 cell lines and 58 primary tumor samples. RESULTS: Silencing expression of GLI1 resulted up-regulation of all target genes in the medulloblastoma cell line, while only PTCH1 was up-regulated in astrocytoma. We also observed methylation of the cyclin D2 promoter in a significant number of astrocytoma cell lines (63%) and primary astrocytoma tumor samples (32%), but not at all in any medulloblastoma samples. PTCH1 promoter methylation was less frequently observed than Cyclin D2 promoter methylation in astrocytomas, and not at all in medulloblastomas. CONCLUSIONS: Our results demonstrate different regulatory mechanisms of Shh-GLI1 signaling. These differences vary according to the downstream target gene affected, the origin of the tissue, as well as epigenetic regulation of some of these genes.

Novel Homeodomain Transcription Factor Nkx2.2 in the Brain Tumor Development.

BACKGROUND: Complex central nervous system (CNS) is made up of neuronal cells and glial cells. Cells of central nervous system are able to regenerate after injury and during repairing. Sonic hedgehog pathway initiated by Shh-N a glycoprotein plays vital role in CNS patterning growth, development and now tumorigenesis. Nkx2.2 homeodomain transcription factor is an effecter molecule, which is positively regulated by Shh during normal growth. Nkx2.2 is essential for V3 domain specification during neural tube patterning at embryonic stage. MBP + oligodendrocytes are differentiated from progenitor cells which express Olig2. Nx2.2 is co-expressed with Olig2 in oligodendrocytes and is essential for later stage of oligodendrocyte maturation. OBJECTIVE: This review paper explores the potential role of Nkx2.2 transcription factor in glioblastoma development. CONCLUSION: Shh pathway plays a vital role in oligodendrocytes differentiation and Nkx2.2 transcription factor is essential for oligodendrocytes differentiation and maturation. Intriguingly, down regulation of Nkx2.2 transcription factor with aberrant Shh signaling pathway is reported in glioma samples. So here it is suggested that Nkx2.2 expression pattern could be used as a potential biomarker for the early diagnosis of glioma.

Exploring the potential role of sonic hedgehog cell signalling pathway in antidepressant effects of nicotine in chronic unpredictable mild stress rat model.

Nicotine is the most common and highly addictive drug of abuse, associated with several life-threatening diseases and high mortality. Nicotine abuse is the concerted effort to feel reward and fight depression in depressed individuals. The underlying mechanism of nicotine is to activate the brain reward system in the central nervous system and provide an antidepressant effect. Antidepressants provide their therapeutic effect by stimulating hippocampal neurogenesis, which can be correlated with brain derived neurotrophic factor (BDNF) expression in the hippocampus. BDNF interacts with Wnt/ß-catenin and sonic hedgehog (Shh) signalling cascade to stimulate hippocampal neurogenesis. Shh is the marker of hippocampal neurogenesis and also involved in the neuropathology of depression. But knowledge in this area to identify the potential therapeutic target is limited. In our study, we explored the role of BDNF, Wnt/ß-catenin and Shh signalling in depression and the involvement of these signalling pathways in providing an antidepressant effect by nicotine. Our investigations showed that chronic unpredictable mild stress induced depression results declined expression of BDNF, Wnt/ß-catenin, Shh and its downstream transcription factors GLI1/2/3 and NKX2.2 in the hippocampus of male Wistar rat. Moreover, we also observed that nicotine administration increased the expression of these signalling molecules in providing the antidepressant effects.

Hedgehog signalling in medulloblastoma, glioblastoma and neuroblastoma.

We investigated a role for Hedgehog signalling in glioblastoma, neuroblastoma and medulloblastoma by studying the transcription of PTCH, SMO, GLI1 and GLI3 in a total of 25 cell lines by standard RT-PCR and qRT-PCR, before and after 5-aza-2'-deoxycytidine and trichostatin A (TSA) treatments. Also 25 glioblastoma samples were tested by qRT-PCR. We also performed real-time methylated specific PCR (qMSP) of the SMO promoter region in DNA from 80 tumor samples (40 glioblastomas and 40 neuroblastomas) and from the 25 cell lines. We detected SMO promoter methylation in more than half of the cell lines and tumor samples. PTCH expression in cell lines was lower than in normal controls, just the opposite to GLI1. SMO and GLI3 expression were high and fully correlated in glioblastoma and medulloblastoma, although partially in neuroblastoma. Our results support the existence of Hedgehog signalling in glioblastoma and medulloblastoma, and to a lesser extent, in neuroblastoma.

The sonic hedgehog-GLI1 signaling pathway in brain tumor development.

INTRODUCTION: The sonic hedgehog (Shh) pathway is a regulatory network involved in development and cancer. Proteins like Ptch, SMO, and Gli are central to the Shh pathway. Other proteins like HHIP, SUFU, Bmi-1, Cyclin D2, Plakoglobin, PAX6, Nkx2.2, and SFRP1 are not so well understood in Shh regulation as Gli-1 downstream target genes. AREAS COVERED: In this review we try to explain the Shh pathway components and their role in development and cancer, mainly of the brain. A summary of each of the proteins is presented together with an overview of their involvement in cancer. EXPERT OPINION: Genetic alterations of the Shh pathway have been detected in cancer stem cells, a subgroup of tumor cells implicated in the origin and maintenance of tumors, being responsible for cancer recurrence and chemotherapy resistance. Cancer stem cells constitute a novel target for biomedical researchers. Specifically, the Shh pathway is being explored as a new opportunity for targeted therapies against tumors. Therefore, a better knowledge of every of the regulators of the Shh pathway is needed.

Role of MXD3 in proliferation of DAOY human medulloblastoma cells.

A subset of medulloblastomas, the most common brain tumor in children, is hypothesized to originate from granule neuron precursors (GNPs) in which the sonic hedgehog (SHH) pathway is over-activated. MXD3, a basic helix-look-helix zipper transcription factor of the MAD family, has been reported to be upregulated during postnatal cerebellar development and to promote GNP proliferation and MYCN expression. Mxd3 is upregulated in mouse models of medulloblastoma as well as in human medulloblastomas. Therefore, we hypothesize that MXD3 plays a role in the cellular events that lead to medulloblastoma biogenesis. In agreement with its proliferative role in GNPs, MXD3 knock-down in DAOY cells resulted in decreased proliferation. Sustained overexpression of MXD3 resulted in decreased cell numbers due to increased apoptosis and cell cycle arrest. Structure-function analysis revealed that the Sin3 interacting domain, the basic domain, and binding to E-boxes are essential for this activity. Microarray-based expression analysis indicated up-regulation of 84 genes and down-regulation of 47 genes. Potential direct MXD3 target genes were identified by ChIP-chip. Our results suggest that MXD3 is necessary for DAOY medulloblastoma cell proliferation. However, increased level and/or duration of MXD3 expression ultimately reduces cell numbers via increased cell death and cell cycle arrest.

Inhibition of the sonic hedgehog pathway by cyplopamine reduces the CD133+/CD15+ cell compartment and the in vitro tumorigenic capability of neuroblastoma cells.

Sonic hedgehog (Hh) developmental pathway deregulation has been proven to play an essential role in several malignancies as neuroblastoma. We found that Hh signaling is active in neuroblastoma, as most pathway components, including GLI1, were expressed in cell lines and tumor samples. Furthermore, SHH ligand expression was found in cell lines and tumors, and GLI1 up-regulation was achieved in response to SHH treatment, suggesting an autocrine mechanism of aberrant activation. A decrease of proliferation and tumorigenic potential, as well as increased apoptosis and a dramatic decrease in the percentage of CD15+ cell population were produced upon Hh inhibition by cyclopamine.