Shh Pathway Genes Research Articles

Novel Porcine Model Reveals Two Distinct LGR5 Cell Types During Lung Development and Homeostasis.

Cells expressing LGR5 play a pivotal role in homeostasis, repair, and regeneration in multiple organs including skin and gastrointestinal tract, yet little is known about their role in the lung. Findings from mice, a widely used animal model, suggest that lung LGR5 expression differs from that of humans. In this work, using a new transgenic pig model, we identify two main populations of LGR5+ cells in the lung that are conserved in human, but not mouse lungs. Using RNA sequencing, 3D imaging and organoid models, we determine that in the fetal lung, epithelial LGR5 expression is transient in a subpopulation of SOX9+/ETV+/SFTPC+ progenitor lung tip cells. In contrast, epithelial LGR5 expression is absent from postnatal lung, but is reactivated in bronchioalveolar organoids derived from basal airway cells. We also describe a separate population of mesenchymal LGR5+ cells that surrounds developing and mature airways, lies adjacent to airway basal cells, and is closely associated with nerve fibers. Transcriptionally, mesenchymal LGR5+ cells include a subset of peribronchial fibroblasts (PBF) that express unique patterns of SHH, FGF, WNT and TGF-β signaling pathway genes. These results support distinct roles for LGR5+ cells in the lung and describe a physiologically relevant animal model for further studies on the function of these cells in repair and regeneration.

Abstract WMP109: Argonaute 2 Regulates Adult Neurogenesis and Oligodendrogenesis After Stroke

Background: Stroke-induced neurogenesis and oligodendrogenesis contribute to improvement of neurological function after stroke. However, mechanisms underlying post stroke neurogenesis and oligodendrogenesis warrant investigation. Argonaute (Ago) genes, the major components of the RNA-induced silencing complex, regulate microRNA (miRNA) function for post-transcriptional gene silencing. The present study investigated the role of neural stem cell (NSC) specific Ago2 after stroke. Methods and Results: Compared to non-stroke NSCs, stroke significantly upregulated Ago2 expression in subventricular zone (SVZ) NSCs. Using adult male mice with conditional ablation of Ago2 in Ascl1 lineage NSCs (Ago2 cKO), we then examined NSC function in neurogenic regions of the SVZ and hippocampus. Under non-ischemic conditions, compared to wild-type littermates (WT), Ago2 cKO mice showed significantly reduced neuronal and oligodendrocyte differentiation of NSCs measured by newly generated neuroblasts (BrdU + /DCX + , 12±7/mm 2 cKO vs 32±11/mm 2 WT, n=3/group, p<0.05) in the neurogenic areas and new mature oligodendrocytes (BrdU + /CC1 + , 20±4/mm 2 cKO vs 40±16/mm 2 WT) in the corpus callosum. In addition, Ago2 cKO mice exhibited the learning and memory impairments. Moreover, Ago2 cKO mice subjected to middle cerebral artery occlusion exhibited significantly reduced sensorimotor functions as measured by the adhesive test, foot-fault test, and modified neurological severity scores compared to WT ischemic mice. Mechanistically, mRNA and miRNA sequencing and bioinformatics analyses showed that ablation of NSC Ago2 deregulated many neurogenic genes involved in the sonic hedgehog (Shh), Notch and TGFβ signaling pathways, and altered Ago2-bound miRNAs that potentially target Shh (miR17-92 cluster), Notch (miR-124, miR-146a) and and TGFβ (miR-21, miR-200c) pathway genes, suggesting that Ago2 regulates neurogenesis and oligodendrogenesis via these miRNA-mRNA interactions. Conclusions: Our data demonstrate an essential role of Ago2 in adult neurogenesis and oligodendrogenesis, and also provide potential therapeutic targets of Ago2-bound miRNAs for improvement of neurological outcomes after stroke by enhancing NSC function.

Coexpression analysis of angiogenesis, proliferation, apoptosis, autophagy and SHH pathway genes involved in skin expansion

Skin and soft tissue expansion is a widely used technique in plastic surgery. However, the regulatory mechanisms associated with cellular processes involved in skin expansion are not well elucidated. In the present study, we aimed at exploring the transcriptome changes associated with skin expansion and profiling the difference in gene expression between the skin tissue in the top of the dilator and the skin tissue in the side of the dilator. A mouse model of skin expansion was established and RNA sequencing (RNA-Seq) was performed on samples collected at different time points. Differential expression analysis was performed using the DESeq2 package while STEM was used for time series clustering profiling. The regulatory networks were established and the functions of sets of genes were analyzed. The mRNA expression levels of candidate genes were validated by the quantitative RT-PCR. Among the skin tissue in the top of the dilator and normal samples at days 1, 3, 7, 14 and 28, 53 commonly upregulated and 7 commonly downregulated genes were identified while among the skin tissue in the side of the dilator and normal samples, 98 downregulated and 255 upregulated genes were identified. Genes differentially expressed among the skin tissue in the top of the dilator and normal samples were involved in coagulation and proliferation-associated pathways while those among the skin tissue in the side of the dilator and normal samples were involved in the inflammation, immune response, and defense response. Among the skin tissue in the top of the dilator and the skin tissue in the side of the dilator samples, 161 were constantly upregulated while 27 were constantly downregulated; these genes were enriched in the biological processes of cell adhesion and regulation of cell proliferation (n = 11). Furthermore, we identified that SHH signaling genes formed a coexpression regulatory network with cellular proliferation, apoptosis, autophagy and angiogenesis-related genes in the expanded skin. In conclusion, our findings can promote research and understanding of the mechanism of skin expansion and will find application in plastic surgery.

MDB-12. TRANS-SPECIES ANALYSIS OF REPLICATION-REPAIR DEFICIENT (RRD) MEDULLOBLASTOMA AND RESPONSE TO IMMUNE-CHECKPOINT INHIBITION: AN IRRDC REPORT

Abstract BACKGROUND Replication-repair deficiency (RRD) stemming from mismatch repair and polymerase-proofreading gene defects (MMRD/PPD) lead to hypermutant childhood cancers, most frequently brain tumors. While medulloblastoma is reported, the clinical, genomic and immune landscape remains unknown. METHODS We analysed the genome, methylome, transcriptome (bulk, single-nuclei) and immune-microenvironment of the largest cohort of RRD-medulloblastoma patients enrolled by the International RRD Consortium, and correlated these to clinical outcomes. RRD mice-models were used to preclinically assess response to immunotherapy. RESULTS RRD-medulloblastoma (n=40) were enriched for anaplasia (55%) and localised disease (85%). Methylation/nano-string-based subgrouping failed to classify 40%, while the remaining clustered with the SHH-subgroup. Copy-number changes were notably infrequent (<20%). All tumors harboured hypermutation and microsatellite instability in contrast to non-RRD controls (p<0.0001). Pathogenic variants were frequent in POLE/POLD1 (80%), TP53 (48%) and SHH-pathway genes (PTCH1, SUFU, SMO: 56%). Interestingly, some tumors additionally had glioma-driver alterations (ATRX, NF1: ~50%), similar to RRD cerebellar glioblastoma, but distinct from both non-RRD medulloblastoma (n=791) and hemispheric glioblastoma (n=733) controls. Moreover, although bulk-transcriptome matched non-RRD SHH-medulloblastoma, single-nuclei analyses suggested an earlier cell-of-origin potentially explaining the heterogenous phenotypes resulting from the driver mutations. Uniquely, immune analyses (gene expression and immunohistochemistry) demonstrated high intra-tumoral CD8 T-cell infiltration. Three-year progression-free survival was 60%. Outcome was worse for RRD-medulloblastoma harbouring TP53-mutation (p=0.04) and failing subgroup-classification (p=0.004). Nestin/Cre MSH2-/- POLE;p.S459F mice developing medulloblastoma recapitulated human disease and demonstrated response to anti-PD1 monotherapy. Recurrent/progressive human tumors including those harbouring TP53-mutations exhibited radiological responses to anti-PD1 monotherapy, which was associated with prolonged ongoing survival in comparison to those not treated with checkpoint-inhibitors (p=0.02). CONCLUSIONS Hypermutant RRD-medulloblastoma reveal heterogenous phenotypes due to unique spectrum of driver mutations and an early cell of origin. Their immune-hot microenvironment allows successful salvage treatment with checkpoint-inhibitors for those failing chemo-radiation, including the high-risk SHH/TP53-mutant and unclassified subgroups.

Abnormal transcriptome-wide DNA demethylation induced by folate deficiency causes neural tube defects.

Neural tube defect (NTDs) is one of the most common and serious fetal and neonatal birth defects. Neural tube closure (NTC) is an exquisitely coordinated process and this procedure is influenced by both genetic and environmental factor. Folic acid (FA) supplementation is an effective for prevention of a proportion of NTDs, however, the mechanism remains unclear. In this study, our data demonstrated genome-wide enrichment of 5-hydroxymethylcytosine (5hmC) modification on active transcriptional start sites (TSS) and decreased 5-methylcytosine (5mC) binding to TSS under folate deficiency in mESCs (mouse embryonic stem cells). Furthermore, folate deficiency promoted 5hmC enrichment enhancer histone 3 lysine 27 acetylation (H3K27ac) binding to Shh pathway genes in mESCs. Upregulation of Shh target genes was observed in mouse brain tissue under low levels of maternal serum folate, along with increased expression of 5-methylcytosine dioxygenase Tet1 levels. Taken together, we found that folate deficiency promoted DNA demethylation and enriched 5hmC through recruitment of H3K27ac to activate the Shh signaling pathway. These results suggest that the 5hmC modification increases concomitantly with a positive correlation to Shh gene expression in folate deficiency-induced mouse NTDs.

HDAC9 structural variants disrupting TWIST1 transcriptional regulation lead to craniofacial and limb malformations.

Structural variants (SVs) can affect protein-coding sequences as well as gene regulatory elements. However, SVs disrupting protein-coding sequences that also function as cis-regulatory elements remain largely uncharacterized. Here, we show that craniosynostosis patients with SVs containing the histone deacetylase 9 (HDAC9) protein-coding sequence are associated with disruption of TWIST1 regulatory elements that reside within the HDAC9 sequence. Based on SVs within the HDAC9‐TWIST1 locus, we defined the 3′-HDAC9 sequence as a critical TWIST1 regulatory region, encompassing craniofacial TWIST1 enhancers and CTCF sites. Deletions of either Twist1 enhancers (eTw5-7Δ/Δ) or CTCF site (CTCF-5Δ/Δ) within the Hdac9 protein-coding sequence led to decreased Twist1 expression and altered anterior/posterior limb expression patterns of SHH pathway genes. This decreased Twist1 expression results in a smaller sized and asymmetric skull and polydactyly that resembles Twist1+/− mouse phenotype. Chromatin conformation analysis revealed that the Twist1 promoter interacts with Hdac9 sequences that encompass Twist1 enhancers and a CTCF site, and that interactions depended on the presence of both regulatory regions. Finally, a large inversion of the entire Hdac9 sequence (Hdac9INV/+) in mice that does not disrupt Hdac9 expression but repositions Twist1 regulatory elements showed decreased Twist1 expression and led to a craniosynostosis-like phenotype and polydactyly. Thus, our study elucidates essential components of TWIST1 transcriptional machinery that reside within the HDAC9 sequence. It suggests that SVs encompassing protein-coding sequences could lead to a phenotype that is not attributed to its protein function but rather to a disruption of the transcriptional regulation of a nearby gene.

Targeted Inhibition of Fibroblast Growth Factor Receptor 1-GLI Through AZD4547 and GANT61 Modulates Breast Cancer Progression.

The underlying mechanism of fibroblast growth factor receptor 1 (FGFR1) mediated carcinogenesis is still not fully understood. For instance, FGFR1 upregulation leads to endocrine therapy resistance in breast cancer patients. The current study aimed to identify FGFR1-linked genes to devise improved therapeutic strategies. RNA-seq and microarray expression data of 1,425 breast cancer patients from two independent cohorts were downloaded for the analysis. Gene Set Enrichment Analysis (GSEA) was performed to identify differentially expressed pathways associated with FGFR1 expression. Validation was done using 150 fresh tumor biopsy samples of breast cancer patients. The clinical relevance of mRNA and protein expression of FGFR1 and its associated genes were also evaluated in mouse embryonic fibroblasts (MEFs) and breast cancer cell line (MDA-MB-231). Furthermore, MDA-MB-231 cell line was treated with AZD4547 and GANT61 to identify the probable role of FGFR1 and its associated genes on cells motility and invasion. According to GSEA results, SHH pathway genes were significantly upregulated in FGFR1 patients in both discovery cohorts of breast cancer. Statistical analyses using both discovery cohorts and 150 fresh biopsy samples revealed strong association of FGFR1 and GLI1, a member of SHH pathway. The increase in the expression of these molecules was associated with poor prognosis, lymph node involvement, late stage, and metastasis. Combined exposures to AZD4547 (FGFR1 inhibitor) and GANT61 (GLI1 inhibitor) significantly reduced cell proliferation, cell motility, and invasion, suggesting molecular crosstalk in breast cancer progression and metastasis. A strong positive feedback mechanism between FGFR1–GLI1 axis was observed, which significantly increased cell proliferation and metastasis. Targeting FGFR1–GLI1 simultaneously will significantly improve the prognosis of breast cancer in patients.

Prenatal alcohol exposure disrupts Sonic hedgehog pathway and primary cilia genes in the mouse neural tube

Neurulation-stage alcohol exposure (NAE; embryonic day [E] 8–10) is associated with midline craniofacial and CNS defects that likely arise from disruption of morphogen pathways, such as Sonic hedgehog (Shh). Notably, midline anomalies are also a hallmark of genetic ciliopathies such as Joubert syndrome. We tested whether NAE alters Shh pathway signaling and the number and function of primary cilia, organelles critical for Shh pathway transduction. Female C57BL/6 J mice were administered two doses of alcohol (2.9 g/kg/dose) or vehicle on E9. Embryos were collected 6, 12, or 24 h later, and changes to Shh, cell cycle genes, and primary cilia were measured in the rostroventral neural tube (RVNT). Within the first 24 h post-NAE, reductions in Shh pathway and cell cycle gene expression and the ratio of Gli3 forms in the full-length activator state were observed. RVNT volume and cell layer width were reduced at 12 h. In addition, altered expression of multiple cilia-related genes was observed at 6 h post-NAE. As a further test of cilia gene-ethanol interaction, mice heterozygous for Kif3a exhibited perturbed behavior during adolescence following NAE compared to vehicle-treated mice, and Kif3a heterozygosity exacerbated the hyperactive effects of NAE on exploratory activity. These data demonstrate that NAE downregulates the Shh pathway in a region of the neural tube that gives rise to alcohol-sensitive brain structures and identifies disruption of primary cilia function, or a “transient ciliopathy”, as a possible cellular mechanism of prenatal alcohol pathogenesis.

Zebrafish Blunt-Force TBI Induces Heterogenous Injury Pathologies That Mimic Human TBI and Responds with Sonic Hedgehog-Dependent Cell Proliferation across the Neuroaxis.

Blunt-force traumatic brain injury (TBI) affects an increasing number of people worldwide as the range of injury severity and heterogeneity of injury pathologies have been recognized. Most current damage models utilize non-regenerative organisms, less common TBI mechanisms (penetrating, chemical, blast), and are limited in scalability of injury severity. We describe a scalable blunt-force TBI model that exhibits a wide range of human clinical pathologies and allows for the study of both injury pathology/progression and mechanisms of regenerative recovery. We modified the Marmarou weight drop model for adult zebrafish, which delivers a scalable injury spanning mild, moderate, and severe phenotypes. Following injury, zebrafish display a wide range of severity-dependent, injury-induced pathologies, including seizures, blood–brain barrier disruption, neuroinflammation, edema, vascular injury, decreased recovery rate, neuronal cell death, sensorimotor difficulties, and cognitive deficits. Injury-induced pathologies rapidly dissipate 4–7 days post-injury as robust cell proliferation is observed across the neuroaxis. In the cerebellum, proliferating nestin:GFP-positive cells originated from the cerebellar crest by 60 h post-injury, which then infiltrated into the granule cell layer and differentiated into neurons. Shh pathway genes increased in expression shortly following injury. Injection of the Shh agonist purmorphamine in undamaged fish induced a significant proliferative response, while the proliferative response was inhibited in injured fish treated with cyclopamine, a Shh antagonist. Collectively, these data demonstrate that a scalable blunt-force TBI to adult zebrafish results in many pathologies similar to human TBI, followed by recovery, and neuronal regeneration in a Shh-dependent manner.

Transcriptional profiling of medulloblastoma with extensive nodularity (MBEN) reveals two clinically relevant tumor subsets with VSNL1 as potent prognostic marker.

Medulloblastoma with extensive nodularity (MBEN) is one of the few central nervous system (CNS) tumor entities occurring in infants which is traditionally associated with good to excellent prognosis. Some MBEN, however, have been reported with an unfavorable clinical course. We performed an integrated DNA/RNA-based molecular analysis of a multi-institutional MBEN cohort (n = 41) to identify molecular events which might be responsible for variability in patients' clinical outcomes. RNA sequencing analysis of this MBEN cohort disclosed two clear transcriptome clusters (TCL) of these CNS tumors: "TCL1 MBEN" and "TCL2 MBEN" which were associated with various gene expression signatures, mutational landscapes and, importantly, prognosis. Thus, the clinically unfavorable "TCL1 MBEN" subset revealed transcriptome signatures composed of cancer-associated signaling pathways and disclosed a high frequency of clinically relevant germline PTCH1/SUFU alterations. In contrast, gene expression profiles of tumors from the clinically favorable "TCL2 MBEN" subgroup were associated with activation of various neurometabolic and neurotransmission signaling pathways, and germline SHH-pathway gene mutations were extremely rare in this transcriptome cluster. "TCL2 MBEN" also revealed strong and ubiquitous expression of VSNL1 (visinin-like protein 1) both at the mRNA and protein level, which was correlated with a favorable clinical course. Thus, combining mutational and epigenetic profiling with transcriptome analysis including VSNL1 immunohistochemistry, MBEN patients could be stratified into clinical risk groups of potential value for subsequent treatment planning.

STEM-18. DISTINCT BUT PREDICTABLE MECHANISMS DRIVE GENETIC VS. EPIGENETIC RESISTANCE TO TARGETED THERAPY

Abstract Emergence of therapy resistance greatly reduces long-term utility of effective targeted therapies, including SMO/SHH pathway inhibitors. SHH signaling is activated in ~25% of human medulloblastomas (MB) and FDA approved SMOi (to treat basal cell carcinoma (BCC)) are currently in clinical trials for MBs and acute myeloid leukemia (AML). Accumulating clinical experience suggests that a significant number of BCC patients treated with SMOi develop acquired resistance over time and some show de novo resistance. A similar pattern is observed in MB patients, indicating the need to elucidate resistance mechanisms, particularly those driving de novo vs. acquired resistance, and develop new strategies to overcome both de novo and acquired resistance to SMOi. We report that we have discovered a novel, epigenetic mechanism of therapy resistance to SMOi that underlie de novo resistance. Using two different mouse models of SHH MB, we tested our original hypothesis that the selective pressure on cancer stem cells (CSCs), but not bulk tumor cells, will determine the resistance mechanism at the molecular level. We show that acquired mutations in the SHH pathway genes (previously reported mechanism of resistance) occur only in tumors that contain CSCs that depend on the SHH pathway. In tumors where only the bulk tumor cells, but not CSCs, depend on SHH signaling, no acquired mutations in the SHH pathway genes are detected. Instead, in these tumors, epigenetic reprogramming through selective degradation of specific histone modifiers results in global changes in the epigenetic cell state and gene expression patterns. Importantly, we can predict the mechanism of resistance in individual tumors prior to treatment based on CSC phenotypes. Finally, we also report biomarkers that can be used to identify tumors with CSCs that are independent of SHH pathway, which can be exploited to design anticipatory combination therapies in the future.

Abstract 1398: Super-enhancers: Possible target in pancreatic cancer for therapeutic approaches

Abstract Introduction: Super enhancers (SE) are distinctive areas of the genome that are densely bound by numerous transcription factors and play a pivotal role in the cell identity, tissue specification, and maintenance. Although the exact functions of super-enhancers are not yet well understood, these regions are known for driving high-level transcription. Studies have established most of the oncogenes playing important role in cancer are driven by SEs. It is likely that the peculiar pancreatic-specific tumor phenotype is a consequence of oncogenes hacking the resident tissue regenerative program, thus interfering with super enhancer-driven repair networks might exert a disproportionately disruptive effect on tumor versus normal pancreatic tissue. The hypothesis of this study is that the tumor cells in PDAC acquire super-enhancers at key oncogenes during tumor development, which drive higher levels of transcription of these genes than in healthy cells. Acquired super-enhancers may thus be biomarkers that could be useful for diagnosis and therapeutic intervention. Thus drugs which could affect super enhancers in cancer may have therapeutic effect. Methods: In this study, we have used ChIP-Seq techniques and bioinformatics analysis following qPCR to identify SE in PDAC cell lines and in pancreatic cancer tissues. Immunohistochemistry, proliferation assays, western blot, RNAseq and CETSA (cellular thermal shift assay) have been performed for understanding the role of enhancers and SE in PDAC. Results: H3K27ac marks were identified at enhancer region of numerous genes that act as SE in PDAC. The most prominent super-enhancers identified, based on a high level of H3K27ac marks were associated with c-MYC, MED1, OCT-4, NANOG and SOX2 genes. Differential association of SE in non-cancerous pancreatic cells, cancerous and metastatic cell lines of PDAC have been implicated in comparison normal and PDAC cells. In this study, we found that GZ17-6.02, combinations of natural compounds, affects acetylation of some of the major SE related genes and at a higher dose, a complete reduction in acetylation marks was seen in embryonic stem cell transcription factors. The mRNA sequencing data after GZ17-6.02 treatment also shows a reduction in transcription of, major transcription factors, SHH pathway genes, and stem cell markers both in vitro and in vivo pancreatic cancer orthotopic models. Cancer cells are more sensitive to lower doses of GZ17-6.02 than normal cell; hence normal cells are expected to have less side effects. Conclusions: There is no systematic study showing an association of SE with pancreatic cancer so far. Hence, in the present study, we have established that several super-enhancer marks can be targeted by combination of natural compounds, GZ17-6.02, in PDAC. This study concludes that super-enhancers can be an important therapeutic target for PDAC. Citation Format: Chandrayee Ghosh, Sumedha Gunewardena, Prasad Dandawate, Santanu Paul, Ossama Tawfik, Cameron West, Shrikant Anant, Animesh Dhar. Super-enhancers: Possible target in pancreatic cancer for therapeutic approaches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1398.

Abstract 165: Cancer stem cell phenotypes determine mechanisms of resistance to targeted therapies

Abstract Despite promising, and sometimes dramatic, efficacy of targeted therapies, primary and acquired resistance to targeted therapies limits their broad and long-term use in the clinic. Therefore, the ability to rationally design anticipatory or combination therapies as early in the treatment process as possible could significantly improve the utility of targeted therapies and cancer outcome. Here, we report that one can predict a priori the types of mutations that will occur in SHH pathway inhibitor treated medulloblastomas based on different cancer stem cell (CSC) phenotypes in each tumor. CSCs are a subset of cancer cells that are more resistant to cytotoxic therapies (chemo and radiation) than bulk tumor cells, and are responsible for tumor recurrence. Previously, we reported that cell-of-origin is a major driver of CSC phenotypes. In particular, we showed that CSCs retain epigenetic memory of their cells-of-origin and the mitogenic pathways that drive CSC proliferation and survival are reflective of their cells-of-origin. We also demonstrated that CSCs and bulk tumor cells do not necessarily depend on the same mitogenic/survival signaling pathways. An important implication of this finding is that targeted therapies selected based on bulk tumor analyses may not be effective in ablating CSCs in some tumors. Furthermore, the selective pressure imposed on CSCs in individual patient tumors by a targeted therapy will vary greatly, depending on whether the selected drug targets a critical pathway in CSCs and not just bulk tumor cells. Another clinically significant implication is that based on the resident CSC phenotype, we may able to predict whether individual SHH tumors will or will not acquire treatment-induced mutations in the SHH pathway upon SHH pathway inhibitors (SMOi) treatment. To test this hypothesis, we treated allografts of spontaneous Ptch;p53 mouse medulloblastomas arising from different cells-of-origins with SMOi's (LDE225 and GDC0449). While all tumors responded to SMOi's initially, most acquired resistance over time. We took an integrated genomics approach to identify molecular mechanisms of therapy resistance in these SMOi-resistant tumors. We report that acquired mutations in SHH pathway genes occurred only in tumors that contained CSCs that depended on the SHH pathway. In tumors where only the bulk tumor cells, but not CSCs, depended on SHH signaling, no acquired mutations in SHH pathway genes were detected. In sum, we report that CSCs drive mechanisms of therapy resistance and also provide the first proof-of-principle evidence that it is possible to predict the types of mutations that will occur in therapy-resistant tumors in advance of treatment, which can be exploited to design anticipatory therapies in the future. Citation Format: Joshy George, Yaohui Chen, Keiko Yamamoto, Scott Adamson, Jeff Chuang, Kyuson Yun. Cancer stem cell phenotypes determine mechanisms of resistance to targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 165.

Multiple roles of Sonic Hedgehog in the developing human cortex are suggested by its widespread distribution

Sonic Hedgehog (Shh) plays an instrumental role in brain development, fine-tuning processes such as cell proliferation, patterning, and fate specification. Although, mutations in the SHH pathway in humans are associated with various neurodevelopmental disorders, ranging from holoprosencephaly to schizophrenia, its expression pattern in the developing human brain is not well established. We now determined the previously not reported wide expression of SHH in the human fetal cerebral cortex during most of the gestation period (10–40 gestational weeks). This spatiotemporal distribution puts Shh in a position to influence the fundamental processes involved in corticogenesis. SHH expression increased during development, shifting from progenitor cells in the proliferative zones to neurons, both glutamatergic and GABAergic, and astrocytes in upper cortical compartments. Importantly, the expression of its downstream effectors and complementary receptors revealed evolutionary differences in SHH-pathway gene expression between humans and rodents.

Characterization of the pleiotropic roles of Sonic Hedgehog during retinal regeneration in adult zebrafish

In contrast to the mammalian retina, the zebrafish retina possesses the ability to regenerate. This is primarily accomplished through Müller glial cells, which, upon damage, re-enter the cell cycle to form retinal progenitors. The progenitors continue to proliferate as they migrate to the area of damage and ultimately differentiate into new neurons. The purpose of this study was to characterize the expression and function of Sonic Hedgehog (Shh) during regeneration of the adult zebrafish retina. Expression profiling of Shh pathway genes showed a significant upregulation of expression associated with stages of progenitor proliferation and neuronal differentiation. Activation of Shh signaling during early stages of retinal regeneration using intraocular injections of the recombinant human SHH (SHH-N) resulted in increased Müller cell gliosis, proliferation, and neuroprotection of damaged retinal neurons. Continued activation of Shh resulted in a greater number of differentiated amacrine and ganglion cells in the fully regenerated retina. Conversely, inhibition of Shh signaling using intraocular injections of cyclopamine resulted in decreased Müller glial cell proliferation and a fewer number of regenerated amacrine and ganglion cells. These data suggest that Shh signaling plays pleiotropic roles in proliferation and differentiation during adult zebrafish retinal regeneration.

Mutations of the Sonic Hedgehog Pathway Underlie Hypothalamic Hamartoma with Gelastic Epilepsy

Hypothalamic hamartoma (HH) with gelastic epilepsy is a well-recognized drug-resistant epilepsy syndrome of early life.(1) Surgical resection allows limited access to the small deep-seated lesions that cause the disease. Here, we report the results of a search for somatic mutations in paired hamartoma- and leukocyte-derived DNA samples from 38 individuals which we conducted by using whole-exome sequencing (WES), chromosomal microarray (CMA), and targeted resequencing (TRS) of candidate genes. Somatic mutations were identified in genes involving regulation of the sonic hedgehog (Shh) pathway in 14/38 individuals (37%). Three individuals had somatic mutations in PRKACA, which encodes a cAMP-dependent protein kinase that acts as a repressor protein in the Shh pathway, and four subjects had somatic mutations in GLI3, an Shh pathway gene associated with HH. In seven other individuals, we identified two recurrent and three single brain-tissue-specific, large copy-number or loss-of-heterozygosity (LOH) variants involving multiple Shh genes, as well as other genes without an obvious biological link to the Shh pathway. The Shh pathway genes in these large somatic lesions include the ligand itself (SHH and IHH), the receptor SMO, and several other Shh downstream pathway members, including CREBBP and GLI2. Taken together, our data implicate perturbation of the Shh pathway in at least 37% of individuals with the HH epilepsy syndrome, consistent with the concept of a developmental pathway brain disease.

Prenatal ethanol exposure in mice phenocopies Cdon mutation by impeding Shh function in the etiology of optic nerve hypoplasia.

ABSTRACTSepto-optic dysplasia (SOD) is a congenital disorder characterized by optic nerve, pituitary and midline brain malformations. The clinical presentation of SOD is highly variable with a poorly understood etiology. The majority of SOD cases are sporadic, but in rare instances inherited mutations have been identified in a small number of transcription factors, some of which regulate the expression of Sonic hedgehog (Shh) during mouse forebrain development. SOD is also associated with young maternal age, suggesting that environmental factors, including alcohol consumption at early stages of pregnancy, might increase the risk of developing this condition. Here, we address the hypothesis that SOD is a multifactorial disorder stemming from interactions between mutations in Shh pathway genes and prenatal ethanol exposure. Mouse embryos with mutations in the Shh co-receptor, Cdon, were treated in utero with ethanol or saline at embryonic day 8 (E8.0) and evaluated for optic nerve hypoplasia (ONH), a prominent feature of SOD. We show that both Cdon−/− mutation and prenatal ethanol exposure independently cause ONH through a similar pathogenic mechanism that involves selective inhibition of Shh signaling in retinal progenitor cells, resulting in their premature cell-cycle arrest, precocious differentiation and failure to properly extend axons to the optic nerve. The ONH phenotype was not exacerbated in Cdon−/− embryos treated with ethanol, suggesting that an intact Shh signaling pathway is required for ethanol to exert its teratogenic effects. These results support a model whereby mutations in Cdon and prenatal ethanol exposure increase SOD risk through spatiotemporal perturbations in Shh signaling activity.

PATTERN OF EXPRESSION OF HUMAN HOMOLOGUE OF DROSOPHILA PATCHED1 PROTEIN IN FOUR DIFFERENT SALIVARY GLAND TUMORS

Salivary gland tumors are a comparatively rare and diverse group of tumors with variable clini- cal and histopathological pictures. Pleomorphic adenoma (PA) & papillary cystadenoma lymphato- sum (PCL) are common benign tumors and Mucoepidermoid carcinoma (MEC) & adenoid cystic carcinoma (ACC) are common malignant tumors with the respect of order. The human homologue of the Drosophila segment polarity gene Patched 1 (PTCH 1) is a member of hedgehog pathway encoding a receptor for the morphogen Sonic Hedgehog and it is the candidate gene for Gorlin’s syndrome. Interestingly it is considered as a one of the tumor a suppressor gene which is associ- ated with different neoplasms and cystic lesions. In this study the pattern of expression of PTCH1 in PA, PCL, MEC, and ACC tumors will be explored to reveal if there is any possible correlation between PTCH1 expression and these four different salivary gland entities. Twenty paraffin embed- ded blocks of salivary gland tumors was chosen as follow (five cases PA, five cases PCL, five cases MEC (two high grades, two low grades, 1 moderate grade) and five cases ACC (4 cribriform pat- tern, one tubular pattern). immunohistochemistry technique using PTCH 1 primary antibody was done to investigate the level of expression of PTCH 1 protein in our samples. Our results revealed that PTCH1 is expressed with various intensities from light to heavy in the four studied salivary gland tumors. Furthermore, PTCH1 expression was confined to the epithelial cells component of the tumors with cytoplasmic and membranous staining. PCL & MEC (low & moderate grade), ACC (cribriform pattern) cases showed the highest immunoreactivity. While PA, high grade MEC, ACC (tubular pattern) showed the lowest. It can be conclude that PTCH1 might has a role in the development of salivary gland tumors with main components epithelium as PCL, MEC, ACC and to a lesser extent PA as a mixed tumor. Finally, it is recommended that researchers should target SHH pathway genes including PTCH1 in most tumors for further investigations and possible gene therapies

Relation between sonic hedgehog pathway gene polymorphisms and basal cell carcinoma development in the Polish population.

In recent decades, increases have been observed in the incidence of nonmelanoma skin cancers, including basal cell carcinoma (BCC) and squamous cell carcinoma. BCC is the most common neoplasm in Caucasian populations. Sonic hedgehog (Shh) pathway impairment plays a key role in BCC pathogenesis, and there is evidence that Shh pathway genetic variations may predispose to BCC development. We genotyped 22 single-nucleotide polymorphisms (SNPs) in 4 Shh pathway genes: SHH, GLI, SMO, and PTCH. The study group consisted of 142 BCC patients and 142 age-matched, sex-matched healthy subjects (controls). SNPs were assessed using the PCR-RFLP method. The genotype distribution for the polymorphisms in the rs104894049 331 A/T SHH, rs104894040 349 T/C SHH, and rs41303402 385 G/A SMO genes differed significantly between the BCC patients and the controls. The presence of CC genotype in the SHH rs104894040 349 T/C polymorphism was linked to the highest risk of BCC development (OR 87.9, p<0.001). Other genotypes, such as the TT in SHH rs104894049 331 A/T and the GG in SMO rs41303402 385 G/A also statistically raised the risk of BCC, but these associations were weaker. Other investigated polymorphisms showed no statistical differences between patients and controls. The results obtained testify to the importance of the SHH and SMO gene polymorphisms in skin cancerogenesis. These results mainly underline the potential role of SHH3 rs104894040 349 T/C gene polymorphism in the development of skin basal cell carcinomas in patients of Polish origin.

Characterization of novel biomarkers in selecting for subtype specific medulloblastoma phenotypes.

Major research efforts have focused on defining cell surface marker profiles for characterization and selection of brain tumor stem/progenitor cells. Medulloblastoma is the most common primary malignant pediatric brain cancer and consists of 4 molecular subgroups: WNT, SHH, Group 3 and Group 4. Given the heterogeneity within and between medulloblastoma variants, surface marker profiles may be subtype-specific. Here, we employed a high throughput flow cytometry screen to identify differentially expressed cell surface markers in self-renewing vs. non-self-renewing SHH medulloblastoma cells. The top 25 markers were reduced to 4, CD271/p75NTR/NGFR, CD106/VCAM1, EGFR and CD171/NCAM-L1, by evaluating transcript levels in SHH tumors relative to samples representing the other variants. However, only CD271/p75NTR/NGFR and CD171/NCAM-L1 maintain differential expression between variants at the protein level. Functional characterization of CD271, a low affinity neurotrophin receptor, in cell lines and primary cultures suggested that CD271 selects for lower self-renewing progenitors or stem cells. Moreover, CD271 levels were negatively correlated with expression of SHH pathway genes. Our study reveals a novel role for CD271 in SHH medulloblastoma and suggests that targeting CD271 pathways could lead to the design of more selective therapies that lessen the broad impact of current treatments on developing nervous systems.