A Pilot Immunohistochemical Study Identifies Hedgehog Pathway Expression in Sinonasal Adenocarcinoma.

Tumors of the head and neck, more specifically the squamous cell carcinoma, often show upregulation of the Hedgehog signaling pathway. However, almost nothing is known about its role in the sinonasal adenocarcinoma, either in intestinal or non-intestinal subtypes. In this work, we have analyzed immunohistochemical staining of six Hedgehog pathway proteins, sonic Hedgehog (SHH), Indian Hedgehog (IHH), Patched1 (PTCH1), Gli family zinc finger 1 (GLI1), Gli family zinc finger 2 (GLI2), and Gli family zinc finger 3 (GLI3), on 21 samples of sinonasal adenocarcinoma and compared them with six colon adenocarcinoma and three salivary gland tumors, as well as with matching healthy tissue, where available. We have detected GLI2 and PTCH1 in the majority of samples and also GLI1 in a subset of samples, while GLI3 and the ligands SHH and IHH were generally not detected. PTCH1 pattern of staining shows an interesting pattern, where healthy samples are mostly positive in the stromal compartment, while the signal shifts to the tumor compartment in tumors. This, taken together with a stronger signal of GLI2 in tumors compared to non-tumor tissues, suggests that the Hedgehog pathway is indeed activated in sinonasal adenocarcinoma. As Hedgehog pathway inhibitors are being tested in combination with other therapies for head and neck squamous cell carcinoma, this could provide a therapeutic option for patients with sinonasal adenocarcinoma as well.

Numb positively regulates Hedgehog signaling at the ciliary pocket.

Hedgehog (Hh) signaling relies on the primary cilium, a cell surface organelle that serves as a signaling hub for the cell. Using proximity labeling and quantitative proteomics, we identify Numb as a ciliary protein that positively regulates Hh signaling. Numb localizes to the ciliary pocket and acts as an endocytic adaptor to incorporate Ptch1 into clathrin-coated vesicles, thereby promoting Ptch1 exit from the cilium, a key step in Hh signaling activation. Numb loss impedes Sonic hedgehog (Shh)-induced Ptch1 exit from the cilium, resulting in reduced Hh signaling. Numb loss in spinal neural progenitors reduces Shh-induced differentiation into cell fates reliant on high Hh activity. Genetic ablation of Numb in the developing cerebellum impairs the proliferation of granule cell precursors, a Hh-dependent process, resulting in reduced cerebellar size. This study highlights Numb as a regulator of ciliary Ptch1 levels during Hh signal activation and demonstrates the key role of ciliary pocket-mediated endocytosis in cell signaling.

Ptch1 is essential for cochlear marginal cell differentiation and stria vascularis formation.

A common cause of deafness in humans is dysregulation of the endocochlear potential generated by the stria vascularis (SV). Thus, proper formation of the SV is critical for hearing. Using single-cell transcriptomics and a series of Shh signaling mutants, we discovered that the Shh receptor Patched1 (Ptch1) is essential for marginal cell (MC) differentiation and SV formation. Single-cell RNA sequencing analyses revealed that the cochlear roof epithelium is already specified into discrete domains with distinctive gene expression profiles at embryonic day 14, with Gsc as a marker gene of the MC lineage. Ptch1 deficiency leads to defective specification of MC precursors along the cochlear basal-apical regions. We demonstrated that elevated Gli2 levels impede MC differentiation through sustaining Otx2 expression and maintaining the progenitor state of MC precursors. Our results uncover an early specification of cochlear non-sensory epithelial cells and establish a crucial role of the Ptch1-Gli2 axis in regulating the development of SV.

Plasma ctDNA Monitoring of a PTCH1-Mutant Metastatic Adult Medulloblastoma Showing a Durable Benefit With Vismodegib.

Adult medulloblastoma (MB) is a rare disease affecting 0.6 persons per million adults over 19 years of age. The SHH-activated/TP53-wild type is the most common subtype, accounting for 60% of adult MBs, being characterized by mutations in PTCH1, SMO, or the TERT promoter. Several small studies demonstrate objective but short-lived responses to SMO inhibitors such as vismodegib or sonidegib. Like other oncogene-addicted solid tumors, detection of the corresponding drivers through liquid biopsy could aid in the molecular diagnosis and monitoring of the disease through less invasive procedures. However, most studies have only evaluated cerebrospinal fluid as the ctDNA reservoir, and very limited evidence exists on the role of liquid biopsy in plasma in patients with primary central nervous system tumors, including MB. We present the case of a 26-year-old patient with a recurrent MB, in which next-generation sequencing (FoundationOne CDx) revealed a mutation in PTCH1, allowing the patient to be treated with vismodegib in second line, resulting in a durable benefit lasting for 1 year. Using an in-house digital PCR probe, the PTCH1 mutation could be tracked in ctDNA during treatment with first-line chemotherapy and while on treatment with vismodegib, demonstrating a precise correlation with the radiological and clinical behavior of the disease.

PTCH1-mutant human cerebellar organoids exhibit altered neural development and recapitulate early medulloblastoma tumorigenesis.

Patched 1 (PTCH1) is the primary receptor for the sonic hedgehog (SHH) ligand and negatively regulates SHH signalling, an essential pathway in human embryogenesis. Loss-of-function mutations in PTCH1 are associated with altered neuronal development and the malignant brain tumour medulloblastoma. As a result of differences between murine and human development, molecular and cellular perturbations that arise from human PTCH1 mutations remain poorly understood. Here, we used cerebellar organoids differentiated from human induced pluripotent stem cells combined with CRISPR/Cas9 gene editing to investigate the earliest molecular and cellular consequences of PTCH1 mutations on human cerebellar development. Our findings demonstrate that developmental mechanisms in cerebellar organoids reflect in vivo processes of regionalisation and SHH signalling, and offer new insights into early pathophysiological events of medulloblastoma tumorigenesis without the use of animal models.

[The molecular genetic aspects of basal cell carcinoma from the position of population health preservation].

The exploration of molecular genetic mechanisms that underlie carcinogenesis, hereditary factors of various oncological diseases, including basal cell carcinoma, the most common type of skin cancer is especially actual and significant for target strategies of public health. The diagnosis of basal cell carcinoma is based on complex clinical, radiologic and genetic examination data. The further research in the field of somatic or hereditary mutations in genes associated with basal cell carcinoma, including Patched 1 (PTCH1), Patched 2 (PTCH2), Smoothed (SMO) continue to be topical. The strategies of primary prevention of basal cell carcinoma, discussions of complex issues of decision-making concerning treatment at primary health care level, training courses and development of guidelines for general practitioners and interdisciplinary recommendations for effective early diagnosis and comprehensive care of basal cell carcinoma are to be suggested.

Methyltransferase-like 3 facilitates the stem cell properties of esophageal cancer by upregulating patched homolog 1 via N6-methyladenosine methylation.

Patched homolog 1 (PTCH1) has been proven to facilitate cell proliferation and self-renewal in esophageal cancer (EC). The present study intended to exploit the influence of PTCH1 on EC cells and the potential mechanisms. PTCH1 and methyltransferase-like 3 (METTL3) expression were examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot in EC cell lines. Following the loss- and gain-of-function assays, cell proliferation was examined by cell counting kit (CCK)-8 and clone formation assays, invasion and migration by Transwell and scratch assays, and the sphere-forming ability of stem cells by cell sphere-forming assay. The expression of stemness genes NANOG homeobox protein (NANOG), octamer-binding transcription factor 4 (Oct4), and sex-determining region Y-box 2 (SOX2) was detected by Western blot. Methylated RNA immunoprecipitation (Me-RIP) assay was performed to test N6-methyladenosine (m6A) modification levels of PTCH1 mRNA, RIP and photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) assays to assess the binding of METTL3 to PTCH1, and actinomycin D treatment to examine PTCH1 mRNA stability. A xenograft tumor model in nude mice was established for further in vivo verification. PTCH1 and METTL3 expression was high in EC cells. Knockdown of METTL3 reduced m6A level and stability of PTCH1 mRNA. Knockdown of PTCH1 or METTL3 declined invasion, proliferation, migration, and NANOG, Oct4, and SOX2 levels in EC cells, and reduced sphere-forming abilities of EC stem cells. Overexpression of PTCH1 abolished the suppressive effect of METTL3 knockdown on EC cells in vitro. METTL3 knockdown repressed tumor growth in nude mice, which was negated by further overexpressing PTCH1. METTL3 facilitated growth and stemness of EC cells via upregulation of PTCH1 expression by enhancing PTCH1 m6A modification.NEW & NOTEWORTHY PTCH1 has been proved to facilitate cell proliferation and self-renewal in esophageal cancer. We studied the upstream regulation mechanism of PTCH1 by METTL3 through m6A modification. Our results provide a new target and theoretical basis for the treatment of esophageal cancer.

Mutations of PTCH1 gene in two pedigrees with bifid rib-basal cell nevus-jaw cyst syndrome. / 肋骨分叉-基底细胞痣-é¢Œéª¨å›Šè‚¿ç»¼åˆå¾ä¸¤å®¶ç³»åŸºå› åºåˆ—å˜å¼‚åˆ†æž.

Two male patients with bifid rib-basal cell nevus-jaw cyst syndrome (BCNS) were admitted to Department of Stomatology, the First Affiliated Hospital of Bengbu Medical College due to radiological findings of multiple low density shadows in the jaw. Clinical and imaging findings showed thoracic malformation, calcification of the tentorium cerebellum and falx cerebrum as well as widening of the orbital distance. Whole exon high-throughput sequencing was performed in two patients and their family members. The heterozygous mutations of c.C2541C>A(p.Y847X) and c.C1501C>T(p.Q501X) in PTCH1 gene were detected in both patients. Diagnosis of BCNS was confirmed. The heterozygous mutations of PTCH1 gene locus were also found in the mothers of the two probands. Proband 1 showed clinical manifestations of low intelligence, and heterozygous mutations of c.C2141T(p.P714L) and c.G3343A(p.V1115I) were detected in FANCD2 gene. Proband 2 had normal intelligence and no FANCD2 mutation. The fenestration decompression and curettage of jaw cyst were performed in both patients. Regular follow-up showed good bone growth at the original lesion, and no recurrence has been observed so far.

Mutations of PTCH1 gene in two pedigrees with bifid rib-basal cell nevus-jaw cyst syndrome / 浙江大学学报·医学版

Two male patients with bifid rib-basal cell nevus-jaw cyst syndrome (BCNS) were admitted to Department of Stomatology, the First Affiliated Hospital of Bengbu Medical College due to radiological findings of multiple low density shadows in the jaw. Clinical and imaging findings showed thoracic malformation, calcification of the tentorium cerebellum and falx cerebrum as well as widening of the orbital distance. Whole exon high-throughput sequencing was performed in two patients and their family members. The heterozygous mutations of c.C2541C>A(p.Y847X) and c.C1501C>T(p.Q501X) in PTCH1 gene were detected in both patients. Diagnosis of BCNS was confirmed. The heterozygous mutations of PTCH1 gene locus were also found in the mothers of the two probands. Proband 1 showed clinical manifestations of low intelligence, and heterozygous mutations of c.C2141T(p.P714L) and c.G3343A(p.V1115I) were detected in FANCD2 gene. Proband 2 had normal intelligence and no FANCD2 mutation. The fenestration decompression and curettage of jaw cyst were performed in both patients. Regular follow-up showed good bone growth at the original lesion, and no recurrence has been observed so far.

New insight into the role of PTCH1 protein in serous ovarian carcinomas.

The Hedgehog (Hh) signaling pathway is essential for normal embryonic development, while its hyperactivation in the adult organism is associated with the development of various cancers. The role of the Hh signaling pathway in ovarian cancer has not been sufficiently investigated. Therefore, the present study investigated the role of protein patched homolog 1 (PTCH1), a component of the Hh signaling pathway, and changes in the promoter methylation status of the corresponding gene in a cohort of low­(LGSC) and high­grade serous ovarian carcinomas (HGSC) and HGSC cell lines (OVCAR8 and OVSAHO). PTCH1 protein expression level was analyzed using immunohistochemistry in tissue samples and immunofluorescence and western blotting in cell lines. DNA methylation patterns of the PTCH1 gene were analyzed using methylation­specific PCR. PTCH1 protein expression was significantly higher in HGSCs and LGSCs compared with controls (healthy ovaries and fallopian tubes). Similarly, ovarian cancer cell lines exhibited significantly higher PTCH1 protein expression compared with a normal fallopian tube non­ciliated epithelial cell line (FNE1). PTCH1 protein fragments of different molecular weights were detected in all cell lines, indicating possible proteolytic cleavage of this protein, resulting in the generation of soluble N­terminal fragments that are translocated to the nucleus. DNA methylation of the PTCH1 gene promoter was exclusively detected in a proportion of HGSC (13.5%) but did not correlate with protein expression. PTCH1 protein was highly expressed in serous ovarian carcinoma tissues and cell lines, while PTCH1 promoter methylation was only detected in HGSC. Further investigation is required to elucidate the possible mechanisms of PTCH1 activation in serous ovarian carcinomas.

Multiple orthokeratinized odontogenic cysts: clinical, pathological, and genetic characteristics.

BACKGROUND: Orthokeratinized odontogenic cyst (OOC) is a rare developmental odontogenic cyst of the jaw. It was originally believed to be a variant of odontogenic keratocyst (OKC) but is now considered to be a distinct entity. OOC usually presents as a single lesion and recurs infrequently. On the other hand, OKC often presents with multiple lesions and displays locally aggressive behavior and a high recurrence rate associated with the protein patched homolog 1 (PTCH1) gene mutation. Multiple OOC cases are extremely rare and seem to be aggressive, but their pathogenesis is not fully understood. This study aimed to determine the clinical, pathological, and genetic characteristics of multiple OCC. METHODS: Three cases of multiple OOC were evaluated for clinical and histological findings, and immunohistochemical expression of Ki-67 and Bcl-2. Furthermore, PTCH1 mutations were analyzed by next-generation sequencing using a custom panel to cover the entire exon of PTCH1. RESULTS: The three cases of multiple OOC included two men and one woman with a mean age of 25.3 years old (range, 18-38 years old). Each case had two or three OOCs (total of seven OOCs), all of which were simultaneously detected. Of the seven OOCs that manifested as multiple jaw cysts, seven (100%) occurred in the posterior regions, four (57.1%) occurred in the mandible, and four (57.1%) were associated with an impacted tooth. Histological examination revealed cysts lined by orthokeratinized stratified squamous epithelium. Immunohistochemistry showed a low Ki-67 labeling index and no Bcl-2 expression in the seven OOCs. No pathogenic PTCH1 mutations were detected in any of the seven OOCs. None of the patients had any other symptoms or signs of recurrence at the last follow-up (6-60 months). CONCLUSION: Multiple OOCs appeared to occur more often in younger patients than solitary OOC. Both multiple and solitary OOCs may be related diseases within the entity of odontogenic cysts. Multiple OOCs are clinicopathologically and genetically distinct from OKC.

Hedgehog Signaling as a Therapeutic Target for Airway Remodeling and Inflammation in Allergic Asthma.

Genome-wide association studies (GWAS) have shown that variants of patched homolog 1 (PTCH1) are associated with lung function abnormalities in the general population. It has also been shown that sonic hedgehog (SHH), an important ligand for PTCH1, is upregulated in the airway epithelium of patients with asthma and is suggested to be involved in airway remodeling. The contribution of hedgehog signaling to airway remodeling and inflammation in asthma is poorly described. To determine the biological role of hedgehog signaling-associated genes in asthma, gene silencing, over-expression, and pharmacologic inhibition studies were conducted after stimulating human airway epithelial cells or not with transforming growth factor ß1 (TGFß1), an important fibrotic mediator in asthmatic airway remodeling that also interacts with SHH pathway. TGFß1 increased hedgehog-signaling-related gene expression including SHH, GLI1 and GLI2. Knockdown of PTCH1 or SMO with siRNA, or use of hedgehog signaling inhibitors, consistently attenuated COL1A1 expression induced by TGFß1 stimulation. In contrast, Ptch1 over-expression augmented TGFß1-induced an increase in COL1A1 and MMP2 gene expression. We also showed an increase in hedgehog-signaling-related gene expression in primary airway epithelial cells from controls and asthmatics at different stages of cellular differentiation. GANT61, an inhibitor of GLI1/2, attenuated TGFß1-induced increase in COL1A1 protein expression in primary airway epithelial cells differentiated in air-liquid interface. Finally, to model airway tissue remodeling in vivo, C57BL/6 wildtype (WT) and Ptch1+/- mice were intranasally challenged with house dust mite (HDM) or phosphate-buffered saline (PBS) control. Ptch1+/- mice showed reduced sub-epithelial collagen expression and serum inflammatory proteins compared to WT mice in response to HDM challenge. In conclusion, TGFß1-induced airway remodeling is partially mediated through the hedgehog signaling pathway via the PTCH1-SMO-GLI axis. The Hedgehog signaling pathway is a promising new potential therapeutic target to alleviate airway tissue remodeling in patients with allergic airways disease.

A392V and R945X mutations cause orofacial clefts via impairing PTCH1 function.

The Hedgehog (HH) signaling plays key roles in embryogenesis and organogenesis, and its dysfunction causes a variety of human birth defects. Orofacial cleft (OFC) is one of the most common congenital craniofacial defects, and its etiology is closely related to mutations in multiple components in the HH pathway, including the PTCH1 receptor. A quantity of PTCH1 variants have been associated with OFC, but the pathogenicity and underlying mechanism of these variants have not been functionally validated. In our previous studies, we identified two PTCH1 variants (A392V and R945X) in two families with hereditary OFC. Here we explore the functional consequences of these two variants. In zebrafish embryos, microinjection of wild type PTCH1 mRNA causes curved body axis and craniofacial anomalies. In contrast, microinjection of A392V and R945X PTCH1 mRNAs results in much milder phenotypes, suggesting these two variants are loss-of-function mutations. In mammalian cells, A392V and R945X mutations reverse the inhibitory effect of PTCH1 on HH signaling. Biochemically, the two mutants PTCH1 show lower expression levels and shortened half-life, indicting these mutations decrease the stability of PTCH1. A392V and R945X mutations also appear to cause PTCH1 to localize away from vesicles. Taken together, our findings indicate that A392V and R945X variants are loss-of-function mutations that disrupt the function of PTCH1 and thus cause dysregulation of HH signaling, leading to the pathogenesis of OFC.

Gorlin Syndrome Associated With a Solitary Circumscribed Retinal Astrocytic Proliferation in a Pediatric Patient.

Gorlin syndrome is a rare autosomal dominant disorder with near complete penetrance. The underlying genetic mechanism is a mutation in a tumor suppressor gene. Thus far, mutations in patched homolog 1 and 2 genes (PTCH1 and PTCH2) and the suppressor of fused gene (SUFU) have been identified. The syndrome is characterized by neoplasms arising early in childhood as well as developmental abnormalities, including ophthalmic anomalies. We present the first case associating Gorlin syndrome with a rare retinal lesion known as solitary circumscribed retinal astrocytic proliferation (SCRAP). SCRAP is a benign, stable retinal tumor. For this reason, it is essential to differentiate it from similar retinal lesions that are associated with poor prognosis. [Ophthalmic Surg Lasers Imaging Retina 2022;53:514-516.].

Long Non-coding RNA SNHG16 Facilitates Esophageal Cancer Cell Proliferation and Self-renewal through the microRNA-802/PTCH1 Axis.

OBJECTIVE: This research sought to explore the effect and mechanism of long non-coding RNA SNHG16 on esophageal cancer (EC) cell proliferation and self-renewal. METHODS: SNHG16 expression was measured in EC9706 and KYSE150 cells. EC9706 and KYSE150 cells were transfected with Lenti-SNHG16, sh-SNHG16, Lenti-protein patched homolog 1 (PTCH1), miR-802 mimic, or miR-802 inhibitor. Flow cytometry was used to sort cancer stem cells (CSCs) in EC9706 and KYSE150 cells. Cell proliferation in EC cells was measured, in addition to colony and tumorsphere numbers. The possible interactions among SNHG16, PTCH1, and miR-802 were identified by dual luciferase reporter and RNA pull-down assays. The expression of the genes in the Hedgehog pathway was detected. Nude mice were injected with SNHG16-silenced EC9706 cells to observe the tumorigenicity of EC9706 cells. RESULTS: Upregulated SNHG16 expression was found in CSCs, whose expression was decreased during the differentiation of CSCs. SNHG16 or PTCH1 overexpression or miR-802 inhibition promoted the proliferation, colony formation, and tumorsphere formation of EC9706 and KYSE150 cells as well as SOX2, OCT4, Bmi-1, and PTCH1 expression. Consistently, SNHG16 knockdown or miR-802 overexpression inhibited EC progression. Moreover, SNHG16 and PTCH1 were competitively bound to miR-802, and SNHG16 orchestrated the miR-802/PTCH1 axis to activate the Hedgehog pathway. SNHG16 silencing repressed the tumorigenicity of EC9706 in nude mice. CONCLUSION: Conclusively, SNHG16 acts as a sponge of miR-802 to upregulate PTCH1 and activate the Hedgehog pathway, thus promoting EC cell proliferation and selfrenewal.

Orthokeratinized odontogenic cysts: A clinicopathologic study of 159 cases and molecular evidence for the absence of PTCH1 mutations.

BACKGROUND: Orthokeratinized odontogenic cyst (OOC), a newly designated entity of odontogenic cysts, is an intraosseous jaw cyst that is entirely or predominantly lined by orthokeratinized squamous epithelium. The aim of this study was to report a large series of OOC to substantiate its clinicopathologic profiles and to investigate PTCH1 mutations in OOCs. METHOD: The clinicopathologic features of 167 OOCs from 159 patients were analyzed and the immunohistochemical expression of markers related to cell differentiation and proliferation was evaluated. Furthermore, PTCH1 mutations were analyzed in 14 fresh samples of OOC. RESULTS: OOCs occurred mostly in the third and fourth decades (60.4%) with a male predilection (66.7%). The lesions developed more often in the mandible than maxilla, primarily in the posterior mandible and ramus. Eight patients (5.0%) showed multiple locations of either bilateral posterior mandible (n = 6) or both the maxilla and mandible. Radiographically, the majority of OOCs (91.2%) showed a well-demarcated, unilocular radiolucency with 14 multilocular cases (8.8%). A follow-up of 131 patients (123 treated by enucleation with or without marsupialization and eight by peripheral ostectomy) revealed no recurrence during an average period of 4.56 years after surgery. Immunohistochemistry indicated lower proliferative activity and a varying epithelial differentiation pattern in OOC compared with odontogenic keratocysts (OKC). No PTCH1 mutation was detected, except for three known single nucleotide polymorphisms. CONCLUSION: The clinicopathological and molecular differences between OOC and OKC justified their separation, and unlike OKCs, OOCs did not harbor PTCH1 mutations, suggesting different pathogenesis underlying these two jaw cysts.

ALKBH5 ameliorated liver fibrosis and suppressed HSCs activation via triggering PTCH1 activation in an m6A dependent manner.

N6-methyladenosine (m6A) is the reversible epigenetic modification of mRNA biogenesis. However, its potential role in HSCs activation and liver fibrosis remains poorly understood. Here we report m6A RNA modification serves as a key layer of HSCs activation and liver fibrosis. The effects of m6A demethylase ALKBH5 on the HSCs activation and liver fibrosis were detected by loss-of-function and gain-of-function analyses. A combination of in vitro and in vivo models, including HSCs and clinical cases or CCl4-induced mice liver fibrosis, was performed to identify the regulation and function of ALKBH5 in liver fibrosis and HSCs activation. Here, we show that the level of ALKBH5 and PTCH1 was decreased in fibrosis livers; however, genetic over expression of LV5-ALKBH5 substantially reduced α-SMA and type I of collagen levels, collagen accumulation, and interstitial fibrosis, while significantly increased PTCH1 levels. Interestingly, the expression of ALKBH5 and PTCH1 was decreased in HSCs treated by TGF-ß1. Moreover, over expression of ALKBH5 reduced HSCs proliferation and migration, whereas ALKBH5 knockdown facilitated HSCs proliferation and migration. Mechanistically, ALKBH5 mediated PTCH1 activation via a m6A-dependent manner. PTCH1 upregulation resulted in the hedgehog signaling inactivation, which inhibited HSCs activation. These findings indicated that ALKBH5 ameliorated liver fibrosis and suppressed HSCs activation via triggering PTCH1 activation in a m6A dependent manner, and provides insight into critical roles of m6A methylation in liver fibrosis.

Impaired Wnt/beta-catenin and protein patched homolog 1 signaling in extraocular sebaceous carcinoma: A clinical and histopathological study.

Sebaceous carcinoma (SC) is a rare malignant neoplasm with sebaceous differentiation. SC is classified into eyelid and extraocular SC clinically. Most studies have focused on the eyelid SC in terms of pathogenesis, treatment, and prognosis. In skin, Wnt/beta-catenin and hedgehog signaling are two major pathways in sebaceous differentiation. We aimed to characterize the clinical and histopathological features of extraocular SC and to measure the expression of beta-catenin, lymphoid enhancer-binding factor 1 (LEF1), sonic hedgehog (Shh), and protein patched homolog 1 (PTCH) in extraocular SC. Ten cases of extraocular SC were identified from 2007 to 2020. The clinical features, microscopic findings, and prognosis were analyzed. Immunohistochemical stain for beta-catenin, LEF1, Shh, and PTCH were performed in extraocular SC and other benign sebaceous tumors including sebaceous hyperplasia, sebaceous adenoma, and sebaceoma. The male:female ratio was 4:6. The median onset age was 73.5 years (range, 43-88). Seven patients out of 10 were diagnosed after 60 years. Most extraocular SC were located on the head and neck with indurated plaque. Two patients had concurrent internal cancers and three patients showed lymph node metastasis at time of presentation. Five-year overall-survival was 40%. Beta-catenin was expressed membranously in all sebaceous hyperplasia, but was expressed variably in extraocular SC (1/5). While LEF1 was unequivocally expressed in normal hair follicles, LEF1 expression was absent in all extraocular SC and benign sebaceous tumors. Regarding the sonic hedgehog signaling, Shh and PTCH were all expressed in the cytoplasm of sebaceous hyperplasia, sebaceous adenoma, and sebaceoma. In contrast, PTCH was absent in all cases of extraocular SC and only 50% of the extraocular SC expressed cytoplasmic Shh. To conclude, extraocular SC commonly affects facial skin in the elderly. Inactivated Wnt/beta-catenin and aberrant hedgehog pathway may contribute to the carcinogenesis of extraocular SC. Further studies may be required to elucidate the causative mechanism of these pathways in extraocular SC.

Differential promoter usages of PTCH1 and down regulation of HHIP are associated with HNSCC progression.

PURPOSE: The study was aimed to understand the importance of the hedgehog signaling pathway in development of head and neck squamous cell carcinoma (HNSCC). METHODS: The molecular profiles of the key regulatory genes of the pathway were analysed in the adjacent normal epithelium and tumor samples. The findings were validated in HNSCC cell line. RESULTS: In the bioinformatical analysis, severe reduction in the expression of HHIP was evident in the datasets. The protein and mRNA expression studies in our sample pool revealed interplay of various isoforms of PTCH1 gene (PTCH1-1 and 1B) together with high/medium expression of GLI, SHH, SMO and HHIP in the basal/parabasal layers of the normal epithelium. As the disease progressed, severe downregulation of HHIP coupled with upregulation of GLI1 and differential expression pattern of various PTCH1 gene isoform was evident. Promoter methylation analysis of PTCH1 gene revealed the involvement of more than one promoter of PTCH1 in regulating the expression of different isoform of this gene during tumorigenesis. Treating the FaDu cell line with the demethylating agent 5-aza-2'-deoxycytidine reversed the methylation effects of HHIP and PTCH1 and de-activated the pathway. Also, reduced expression of HHIP-AS1 was observed in our sample pool suggesting multiple ways of regulation of the HHIP gene. Lastly, the patients with under expression of HHIP, HHIP-AS1, high expression of GLI1 showed worse five-year over-all survival trend. CONCLUSION: Dynamic promoter switching of PTCH1 and frequent inactivation of HHIP are the key regulatory events of hedgehog pathway activation in HNSCC.

Structural basis for catalyzed assembly of the Sonic hedgehog-Patched1 signaling complex.

The dually lipidated Sonic hedgehog (SHH) morphogen signals through the tumor suppressor membrane protein Patched1 (PTCH1) to activate the Hedgehog pathway, which is fundamental in development and cancer. SHH engagement with PTCH1 requires the GAS1 coreceptor, but the mechanism is unknown. We demonstrate a unique role for GAS1, catalyzing SHH-PTCH1 complex assembly in vertebrate cells by direct SHH transfer from the extracellular SCUBE2 carrier to PTCH1. Structure of the GAS1-SHH-PTCH1 transition state identifies how GAS1 recognizes the SHH palmitate and cholesterol modifications in modular fashion and how it facilitates lipid-dependent SHH handoff to PTCH1. Structure-guided experiments elucidate SHH movement from SCUBE2 to PTCH1, explain disease mutations, and demonstrate that SHH-induced PTCH1 dimerization causes its internalization from the cell surface. These results define how the signaling-competent SHH-PTCH1 complex assembles, the key step triggering the Hedgehog pathway, and provide a paradigm for understanding morphogen reception and its regulation.