Modeling SHH-driven medulloblastoma with patient iPS cell-derived neural stem cells.

Medulloblastoma is the most common malignant brain tumor in children. Here we describe a medulloblastoma model using Induced pluripotent stem (iPS) cell-derived human neuroepithelial stem (NES) cells generated from a Gorlin syndrome patient carrying a germline mutation in the sonic hedgehog (SHH) receptor PTCH1. We found that Gorlin NES cells formed tumors in mouse cerebellum mimicking human medulloblastoma. Retransplantation of tumor-isolated NES (tNES) cells resulted in accelerated tumor formation, cells with reduced growth factor dependency, enhanced neurosphere formation in vitro, and increased sensitivity to Vismodegib. Using our model, we identified LGALS1 to be a GLI target gene that is up-regulated in both Gorlin tNES cells and SHH-subgroup of medulloblastoma patients. Taken together, we demonstrate that NES cells derived from Gorlin patients can be used as a resource to model medulloblastoma initiation and progression and to identify putative targets.

Metastatic Basal Cell Carcinoma Arising From a Primary Cutaneous Carcinosarcoma.

Primary cutaneous carcinosarcoma is a rare malignant tumor composed of both an epithelial and mesenchymal cell population. We present a case of a man, aged 56 years, found to have a 26-mm exophytic lesion on the vertex scalp identified to contain a distinct population of basal cell carcinoma (BCC) as well as another population of spindled cells representing a poorly differentiated sarcomatous component. Five years after the removal of the primary lesion, the patient presented with metastatic BCC to the right scalp, right cervical nodes, lung, and rib. Next-generation sequencing of the lung metastasis was performed, revealing mutation of the patched gene (PTCH1) and prompting treatment with vismodegib (Erivedge). Cases of primary cutaneous carcinosarcoma with a basal cell epithelial component are rare and not much is known about their pathogenesis or clinical course. This case is unique in that metastatic BCC arose from a primary carcinosarcoma in which the carcinomatous component was basal cell. Furthermore, it has clinical significance in the successful use of a selective hedgehog pathway inhibitor.

Cutaneous Keratocyst With D2-40 Immunoreactivity in Basal Cell Nevus Syndrome.

ABSTRACT: Although not a diagnostic criterion for basal cell nevus syndrome (BCNS, OMIM#109400), cutaneous cysts, particularly epidermoid cysts, are common in this condition. Cutaneous keratocysts, on the other hand, are extremely rare in general and have been identified in only 5 patients with BCNS. Here, we describe a BCNS patient with a cutaneous keratocyst that demonstrated D2-40 (podoplanin) immunoreactivity, which has been detected in odontogenic keratocysts but not cutaneous keratocysts. This finding suggests that cutaneous keratocysts may be developmentally homologous to odontogenic keratocysts and may behave similarly in terms of invasion and growth pattern.

Keratinocytes from Gorlin Syndrome-induced pluripotent stem cells are resistant against UV radiation.

Gorlin syndrome (GS) is an autosomal dominant genetic disorder involving Patched 1 (PTCH1) mutations. The PTCH1 is a receptor as well as an inhibitor of hedgehog (Hh) to sequester downstream Hh pathway molecules called Smoothened (SMO). PTCH1 mutations causes a variety of GS conditions including falx calcification, odontogenic keratocytes and basal cell carcinomas (BCC). Because PTCH1 is a major driver gene of sporadic BCC, GS patients are characteristically prone to BCC. In order to elucidate the pathological mechanism of BCC-prone GS patients, we investigated keratinocytes derived from GS patient specific iPS cells (G-OFiPSCs) which were generated and reported previously. We found that keratinocytes derived from G-OFiPSCs (GKCs) have increased expression of Hh target molecules. GKCs were irradiated and those cells showed high resistance to UV induced apoptosis. BCL2, known as anti-apoptotic molecule as well as Hh target, significantly increased in GKCs. Several molecules involved in DNA repair, cell cycle control, senescence, and genotoxic stress such as TP53, BRCA1 and GADD45A increased only in GKCs. GKCs are indicated to be resistant to UV irradiation by upregulating molecules which control DNA repair and genotoxic even under DNA damage caused by UV. The anti-apoptotic properties of GKCs may contribute BCC.

Establishment of a Gorlin syndrome model from induced neural progenitor cells exhibiting constitutive GLI1 expression and high sensitivity to inhibition by smoothened (SMO).

The hedgehog signaling pathway is a vital factor for embryonic development and stem cell maintenance. Dysregulation of its function results in tumor initiation and progression. The aim of this research was to establish a disease model of hedgehog-related tumorigenesis with Gorlin syndrome-derived induced pluripotent stem cells (GS-iPSCs). Induced neural progenitor cells from GS-iPSCs (GS-NPCs) show constitutive high GLI1 expression and higher sensitivity to smoothened (SMO) inhibition compared with wild-type induced neural progenitor cells (WT-NPCs). The differentiation process from iPSCs to NPCs may have similarity in gene expression to Hedgehog signal-related carcinogenesis. Therefore, GS-NPCs may be useful for screening compounds to find effective drugs to control Hedgehog signaling activity.

Gorlin Syndrome: Recent Advances in Genetic Testing and Molecular and Cellular Biological Research.

Gorlin syndrome is a skeletal disorder caused by a gain of function mutation in Hedgehog (Hh) signaling. The Hh family comprises of many signaling mediators, which, through complex mechanisms, play several important roles in various stages of development. The Hh information pathway is essential for bone tissue development. It is also the major driver gene in the development of basal cell carcinoma and medulloblastoma. In this review, we first present the recent advances in Gorlin syndrome research, in particular, the signaling mediators of the Hh pathway and their functions at the genetic level. Then, we discuss the phenotypes of mutant mice and Hh signaling-related molecules in humans revealed by studies using induced pluripotent stem cells.

The Role of Dermal Fibroblasts in Nevoid Basal Cell Carcinoma Syndrome Patients: An Overview.

Nevoid basal cell carcinoma syndrome (NBCCS), also named Gorlin syndrome, is a rare multisystem genetic disorder characterized by marked predisposition to basal cell carcinomas (BCCs), childhood medulloblastomas, maxillary keratocysts, celebral calcifications, in addition to various skeletal and soft tissue developmental abnormalities. Mutations in the tumor suppressor gene PATCHED1 (PTCH1) have been found to be associated in the majority of NBCCS cases. PATCH1 somatic mutations and loss of heterozygosity are also very frequent in sporadic BCCs. Unlike non-syndromic patients, NBCCS patients develop multiple BCCs in sun-protected skin area starting from early adulthood. Recent studies suggest that dermo/epidermal interaction could be implicated in BCC predisposition. According to this idea, NBCCS fibroblasts, sharing with keratinocytes the same PTCH1 germline mutation and consequent constitutive activation of the Hh pathway, display features of carcinoma-associated fibroblasts (CAF). This phenotypic traits include the overexpression of growth factors, specific microRNAs profile, modification of extracellular matrix and basement membrane composition, increased cytokines and pro-angiogenic factors secretion, and a complex alteration of the Wnt/-catenin pathway. Here, we review studies about the involvement of dermal fibroblasts in BCC predisposition of Gorlin syndrome patients. Further, we matched the emerged NBCCS fibroblast profile to those of CAF to compare the impact of cell autonomous "pre-activated state" due to PTCH1 mutations to those of skin tumor stroma.

Immunohistochemical evaluation of Sonic Hedgehog signaling pathway proteins (Shh, Ptch1, Ptch2, Smo, Gli1, Gli2, and Gli3) in sporadic and syndromic odontogenic keratocysts.

AIMS: The aim of this study was to compare the clinical and demographic features of 62 patients presenting sporadic odontogenic keratocysts (OKCs) or OKCs associated with nevoid basal cell carcinoma syndrome (NBCCS). In conjunction with this, we also evaluated the immunohistochemical expression of Shh, Ptch1, Ptch2, Smo, Gli1, Gli2 and Gli3 proteins in 86 OKCs. By doing this, we add to the understanding of the biology of this type of lesion, providing tools that will help facilitate the early diagnosis of NBCCS in those patients where the first manifestation is that of OKCs. METHODS: This is a retrospective study; patients were classified into two groups: group 1 which consisted of those who were not affected by NBCCS (49 patients and 57 OKCs) and group 2 which consisted of those who were diagnosed with NBCCS (13 patients and 29 OKCs). The clinical and demographic features were studied and the immunohistochemical expression of Sonic Hedgehog proteins (Shh, Ptch1, Ptch2, Smo, Gli1, Gli2, and Gli3) was analyzed in all samples. RESULTS: There was an increase in the expression of three proteins in the syndromic OKC, when compared to that of sporadic cysts. Shh and Gli1 showed higher cytoplasmic expression, while Smo revealed stronger nuclear and cytoplasmic expressions. CONCLUSION AND CLINICAL RELEVANCE: Our findings suggest that the expression patterns of important Shh pathway proteins can represent valuable markers for early diagnosis of NBCCS-associated OKCs, as the major criterion for the diagnosis of NBCCS is currently based on the late appearance of basal cellular carcinomas. Thus, standardizing a new diagnostic tool for diagnosis of NBCCS could be of great importance in the identification of therapeutic targets. We therefore suggest, as based on our findings, that OKCs showing high expression of Shh, Smo, and Gli1 are potentially associated with NBCCS.

PTCH1 isoform 1b is the major transcript in the development of basal cell nevus syndrome.

Basal cell nevus syndrome (BCNS) is an autosomal dominant disorder most commonly caused by a germline mutation in the PTCH1 gene. PTCH1 is known to have different isoforms with different functional properties and expression patterns among tissues. We detected a novel, pathogenic de novo mutation in PTCH1 isoform 1b (c.114delG) in a BCNS patient. Furthermore, we elucidated the specific expression pattern of PTCH1 isoforms in normal skin, BCC and peripheral blood by studying expression of different PTCH1 isoforms. Human skin showed expression of isoforms 1b and 1d, while peripheral blood additionally showed 1a and 1e expression. BCCs showed expression of all isoforms. Here we report a patient with a novel, isoform 1b specific mutation in PTCH1 and thereby distinguish PTCH1 isoform 1b as the major transcript in the development of BCNS.

Brain morphology in children with nevoid basal cell carcinoma syndrome.

Brain morphology is tightly regulated by diverse signaling pathways. Hedgehog signaling is a candidate pathway considered responsible for regulating brain morphology. Nevoid basal cell carcinoma syndrome (NBCCS), caused by a PTCH1 mutation in the hedgehog signaling pathway, occasionally exhibits macrocephaly and medulloblastoma. Although cerebellar enlargement occurs in ptch1 heterozygous-deficient mice, its impact on human brain development remains unknown. We investigated the brain morphological characteristics of children with NBCCS. We evaluated brain T1-weighted images from nine children with NBCCS and 15 age-matched normal control (NC) children (mean [standard deviation], 12.2 [2.8] vs. 11.6 [2.3] years old). The diameters of the cerebrum, corpus callosum, and brain stem and the cerebellar volume were compared using two-tailed t-tests with Welch's correction. The transverse diameters (150.4 [9.9] vs. 136.0 [5.5] mm, P = 0.002) and longitudinal diameters (165.4 [8.0] vs. 151.3 [8.7] mm, P = 0.0007) of the cerebrum, cross-sectional area of the cerebellar vermis (18.7 [2.6] vs. 11.8 [1.7] cm2 , P = 0.0001), and total volume of the cerebellar hemispheres (185.1 [13.0] vs. 131.9 [10.4] cm3 , P = 0.0001) were significantly larger in the children with NBCCS than in NC children. Thinning of the corpus callosum and ventricular enlargement were also confirmed in children with NBCCS. We demonstrate that, on examination of the brain morphology, an increase in the size of the cerebrum, cerebellum, and cerebral ventricles is revealed in children with NBCCS compared to NC children. This suggests that constitutively active hedgehog signaling affects human brain morphology and the PI3K/AKT and RAS/MAPK pathways.

Hedgehog signaling is synergistically enhanced by nutritional deprivation and ligand stimulation in human fibroblasts of Gorlin syndrome.

Hedgehog signaling is a pivotal developmental pathway that comprises hedgehog, PTCH1, SMO, and GLI proteins. Mutations in PTCH1 are responsible for Gorlin syndrome, which is characterized by developmental defects and tumorigenicity. Although the hedgehog pathway has been investigated extensively in Drosophila and mice, its functional roles have not yet been determined in human cells. In order to elucidate the mechanism by which transduction of the hedgehog signal is regulated in human tissues, we employed human fibroblasts derived from three Gorlin syndrome patients and normal controls. We investigated GLI1 transcription, downstream of hedgehog signaling, to assess native signal transduction, and then treated fibroblasts with a recombinant human hedgehog protein with or without serum deprivation. We also examined the transcriptional levels of hedgehog-related genes under these conditions. The expression of GLI1 mRNA was significantly higher in Gorlin syndrome-derived fibroblasts than in control cells. Hedgehog stimulation and nutritional deprivation synergistically enhanced GLI1 transcription levels, and this was blocked more efficiently by vismodegib, a SMO inhibitor, than by the natural compound, cyclopamine. Messenger RNA profiling revealed the increased expression of Wnt signaling and morphogenetic molecules in these fibroblasts. These results indicated that the hedgehog stimulation and nutritional deprivation synergistically activated the hedgehog signaling pathway in Gorlin syndrome fibroblasts, and this was associated with increments in the transcription levels of hedgehog-related genes such as those involved in Wnt signaling. These fibroblasts may become a significant tool for predicting the efficacies of hedgehog molecular-targeted therapies such as vismodegib.

Basal cell carcinoma of the skin (part 1): epidemiology, pathology and genetic syndromes.

Basal cell carcinoma (BCC) is the most common skin cancer worldwide with increasing incidence, but difficult to assess due to the current under registration practice. Despite the low mortality rate, BCC is a cause of great morbidity and an economic burden to health services. There are several risk factors that increase the risk of BCC and partly explain its incidence. Low-penetrance susceptibility alleles, as well as genetic alterations in signaling pathways, namely SHH pathway, also contribute to the carcinogenesis. BCC associate with several genetic syndromes, of which basal cell nevus syndrome is the most common.

Immunohistochemical analysis of matrix metalloproteinases (1, 2, and 9), Ki-67, and myofibroblasts in keratocystic odontogenic tumors and pericoronal follicles.

BACKGROUND: Keratocystic odontogenic tumor (KCOT) is a benign tumor that arises sporadically or associated with nevoid basal cell carcinoma syndrome (NBCCS). Its locally aggressive behavior contrasts with its cystic histological appearance. To better understand the interaction between tumor cells and the stroma, the present study aimed to evaluate and compare the immunohistochemical expression of matrix metalloproteinases (MMP-1, -2, and -9), the cellular proliferation index (Ki-67), and the presence of myofibroblasts (MFs) in KCOTs. METHODS: Eleven cases of isolated KCOT (G1) and 12 cases of KCOT associated with NBCCS (G2) were selected for an immunohistochemical investigation of the proteins MMP-1, MMP-2, MMP-9, Ki-67, and α-smooth muscle actin (α-SMA) in MFs. A group of 6 pericoronal follicles (G3) was included as a normal odontogenic tissue control. RESULTS: Significant differences between the G3 and G1/G2 groups regarding the expression of MMP-1, MMP-9 (in connective tissue), and Ki-67 were observed. In KCOT, there was a positive correlation between the Ki-67 antigen and MMP-1 and between MFs and MMP-1 in the parenchyma. No statistical differences were found between G1 and G2 groups. CONCLUSIONS: MMP-1, MMP-9, and proliferative activity appear to play important roles in KCOT pathogenesis. The increased proliferative activity with KCOT was associated with elevated MMP-1 production in the parenchyma, which influenced the growth of the lesion in association with an increased number of MFs.

Prognostic relevance of clinicopathological factors in sporadic and syndromic odontogenic keratocysts: A comparative study.

BACKGROUND: Current evidence suggests that nevoid basal cell carcinoma syndrome (NBCCS)-associated odontogenic keratocysts (OKCs) exhibit more aggressive clinical behavior and a higher tendency to relapse. The prognostic efficacy of various markers in sporadic and syndromic OKCs is unclear, and so are the results of studies on the usefulness of immunohistochemistry in distinguishing syndromic from sporadic OKCs. OBJECTIVES: This retrospective study aimed to compare the prognostic relevance of various clinicoradiological and histopathological features, as well as the immunoexpression of COX-2, Bcl-2, proliferating cell nuclear antigen (PCNA), p53, Ki-67, osteoprotegerin (OPG), receptor activator of nuclear factor κ B (RANK) and receptor activator of nuclear factor κ B ligand (RANKL), as well as RANKL/OPG balance between sporadic and syndromic OKCs, and to test their utility in distinguishing the 2 types of OKC. MATERIAL AND METHODS: We compared the immunoexpression of the aforementioned markers between 31 sporadic and 12 syndromic OKCs, and tested clinicopathological findings and levels of immunostaining against recurrence. RESULTS: We found a significant association between NBCCS and OKC recurrence. There were significant differences in PCNA, p53 and OPG immunoexpression between sporadic and syndromic OKCs. We also found that recurrent sporadic OKCs were significantly larger and markedly more often associated with cortical perforation. Recurrent sporadic OKCs exhibited COX-2 upregulation, but we failed to demonstrate its prognostic relevance. Recurrent syndromic OKCs showed a markedly higher RANKL > OPG ratio. CONCLUSIONS: The NBCCS-associated OKCs are significantly more prone to recur than their sporadic counterparts. Larger size and radiological signs of cortical perforation in sporadic OKCs may indicate a higher risk of recurrence. The COX-2 is upregulated in recurrent sporadic OKCs, whereas recurrent syndromic OKCs exhibit higher RANKL and lower OPG expression; however, these findings have no prognostic relevance. The immunoexpression of p53, PCNA and OPG may help to distinguish syndromic from sporadic OKCs.

Selective haploinsufficiency of longer isoforms of PTCH1 protein can cause nevoid basal cell carcinoma syndrome.

Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterized by developmental defects and tumorigenesis. The gene responsible for NBCCS is PTCH1. The PTCH1 gene has five alternatively used first exons resulting in the translation of three isoforms of the PTCH1 protein; that is, PTCHL, PTCHM and PTCHS. However, the biological significance of each isoform is unclear. Here we show an individual with NBCCS carrying a nonsense mutation in PTCH1 exon2, c.387G>A (p.W129X). As the mutation lay upstream of the ATG codon used for PTCHS translation, the mutant allele still expressed RNA isoforms that encode PTCHS. These results clearly demonstrate that a selective haploinsufficiency of longer isoforms of the PTCH1 protein, PTCHL and PTCHM, but not PTCHS is sufficient to cause NBCCS. Although mice selectively deficient in PTCHS isoforms are currently unavailable, this study sheds light on the complex in vivo roles of PTCH1 isoforms.

Primitive neuroectodermal tumors of the central nervous system associated with genetic and metabolic defects.

AIM: To evaluate the genetic, congenital and metabolic disorders which were detected concurrently with primitive neuroectodermal tumors (PNET) of the central nervous system in children. METHODS: Medical records of 1030 children who were admitted to our department with diagnosis of brain tumor between 1975 and 2005 were reviewed retrospectively. Medulloblastoma and supratentorial PNETs were detected in 289 patients. They were reviewed for associated metabolic conditions, genetic and congenital defects. RESULTS: One of the following conditions were detected in 10 patients with medulloblastoma and supratentorial PNETs: Neurofibromatosis type 1, Gorlin syndrome, juvenile polyposis coli, cancer prone syndrome of total premature chromatid separation and Fanconi anemia, bilateral retinoblastoma, L-2-hydroxyglutaric aciduria, Gilbert syndrome, gray platelet syndrome, cleft lip-palate and left renal agenesis. In the patients with multiple malignant diseases, cancer prone syndrome of total premature chromatid separation and Fanconi anemia, Gorlin syndrome and juvenile polyposis coli were diagnosed after diagnosis of the malignant tumors. Medulloblastoma was the first manifestation in the case with Gorlin syndrome. In case with retinoblastoma, pineal PNET was detected 2 months after diagnosis of retinoblastoma. Cleft lip-palate and L-2-Hydroxyglutaric aciduria were detected previously in the patients before their brain tumors whereas Gray platelet, Gilbert syndrome and left renal agenesis were diagnosed during treatment of medulloblastoma. CONCLUSION: Associated genetic, metabolic and congenital conditions were detected in 3.5% of the cases. Thus the patients with PNET should be followed for these defects.

Distinct non-clock-like signatures of the basal cell carcinomas from three sisters with a lethal Gorlin-Goltz syndrome.

BACKGROUND: Gorlin-Goltz syndrome (GS) is an inherited disease characterized by predisposition to basal cell carcinomas (BCCs) and various developmental defects, whose numerous disease-causing PTCH1 mutations have been identified in the hedgehog (Hh) signaling pathway. METHODS: In this study, whole exome sequencing was used to screen for both somatic and germline deleterious mutations in three sisters with a lethal GS. The mutations we found were confirmed by subcloning and Sanger sequencing of the genomic DNA. RNA-seq was performed to profile gene expression in paired BCCs samples and the expression levels for selected genes were validated by quantitative PCR. RESULTS: The clinical and histopathologic features were analyzed for the proband in the three-generation GS family. We identified the insertion mutation PTCH1 c.1341dupA (p. L448Tfs*49), which segregated with BCC phenotype and contributed to the death of two in four patients from a Chinese family with GS. Compared with adjacent non-cancerous tissues (ANCT), four second-hit mutations were found in four of the six pairs of BCC from three patients. Of note, somatic genomic alterations in all six BCC samples were mainly clustered into non-clock-like Signature 7 (ultraviolet mutagenesis) and 11 (related to certain alkylating agents). Both RNA-seq and quantitative RT-PCR confirmed that the mRNA levels of PTCH1 and its effector GLI1 were markedly upregulated in six pairs of BCC samples versus ANCT. CONCLUSIONS: The distinct non-clock-like signatures of BCCs indicated that GS was not a life-threatening illness. The main reasons for untimely death of GS patients were PTCH1 mutation, exposure to intense ultraviolet radiationand the poor economic conditions.

Expression of Matrix Metalloproteinases 7 and 9, Desmin, Alpha-Smooth Muscle Actin and Caldesmon, in Odontogenic Keratocyst Associated with NBCCS, Recurrent and Sporadic Keratocysts.

Nevoid basal cell carcinoma syndrome (NBCCS) associated odontogenic keratocysts (OKCs) show more aggressive behavior and it has a higher frequency of relapse than non-syndromic OKCs. Stromal myofibroblasts (MFs), characterized by α-smooth muscle actin (αSMA), desmin and caldesmon expression, and metalloproteinases (MMPs) have an essential role in the remodeling of the extracellular matrix (ECM). The aim of the study is to analyze the immunohistochemical expression of MMP-7, MMP-9, αSMA and other new markers in the study of OKCs MFs such as desmin and caldesmon in NBCCS-associated OKCs compared to recurrent and sporadic keratocysts. Fourty 40 patients (23 M and 17 F) underwent surgery to remove the OKCs. The histological sections in paraffin were incubated with markers antibodies and a semi-quantitative score was used to evaluate the immunoreactivity. Densitometric analysis showed a very significantly increased expression of αSMA, caldesmon, MMP-7 and MMP-9 in NBCCS-OKCs compared to non-syndromic OKCs (p < 0.001). However, desmin showed a not significant increased expression in non-syndromic OKC compared to NBCCS-OKCs specimens in which desmin was slightly or not at all expressed. NBCSS-OKCs showed a greater distribution of MFs compared to the other OKCs subtypes. Further studies will be needed to evaluate whether the different expressions of these markers can be correlated to a different clinical behavior.

Genetic elimination of Suppressor of fused reveals an essential repressor function in the mammalian Hedgehog signaling pathway.

The Hedgehog (Hh) pathway plays important roles during embryogenesis and carcinogenesis. Here, we show that ablation of the mouse Suppressor of fused (Sufu), an intracellular pathway component, leads to embryonic lethality at approximately E9.5 with cephalic and neural tube defects. Fibroblasts derived from Sufu(-/-) embryos showed high Gli-mediated Hh pathway activity that could not be modulated at the level of Smoothened and could only partially be blocked by PKA activation. Despite the robust constitutive pathway activation in the Sufu(-/-) fibroblasts, the GLI1 steady-state localization remained largely cytoplasmic, implying the presence of an effective nuclear export mechanism. Sufu(+/-) mice develop a skin phenotype with basaloid changes and jaw keratocysts, characteristic features of Gorlin syndrome, a human genetic disease linked to enhanced Hh signaling. Our data demonstrate that, in striking contrast to Drosophila, in mammals, Sufu has a central role, and its loss of function leads to potent ligand-independent activation of the Hh pathway.