Peter G. Zaphiropoulos

Mutations of the Human Homolog of Drosophila patched in the Nevoid Basal Cell Carcinoma Syndrome

The nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterized by multiple basal cell carcinomas (BCCs), pits of the palms and soles, jaw keratocysts, a variety of other tumors, and developmental abnormalities. NBCCS maps to chromosome 9q22.3. Familial and sporadic BCCs display loss of heterozygosity in this region, consistent with the gene being a tumor suppressor. A human sequence (PTC) with strong homology to the Drosophila segment polarity gene, patched, was isolated from a YAC and cosmid contig of the NBCCS region. Mutation analysis revealed alterations of PTC in NBCCS patients and in related tumors. We propose that a reduction in expression of the patched gene can lead to the developmental abnormalities observed in the syndrome and that complete loss of patched function contributes to transformation of certain cell types.

Mammalian Suppressor-of-Fused modulates nuclear–cytoplasmic shuttling of GLI-1

Sonic hedgehog, Patched and Gli are components of a mammalian signalling pathway that has been conserved during evolution and which has a central role in the control of pattern formation and cellular proliferation during development. Here we identify the human Suppressor-of-Fused (SUFUH) complementary DNA and show that the gene product interacts physically with the transcriptional effector GLI-1, can sequester GLI-1 in the cytoplasm, but can also interact with GLI-1 on DNA. Functionally, SUFUH inhibits transcriptional activation by GLI-1, as well as osteogenic differentiation in response to signalling from Sonic hedgehog. Localization of GLI-1 is influenced by the presence of a nuclear-export signal, and GLI-1 becomes constitutively nuclear when this signal is mutated or nuclear export is inhibited. These results show that SUFUH is a conserved negative regulator of GLI-1 signalling that may affect nuclear–cytoplasmic shuttling of GLI-1 or the activity of GLI-1 in the nucleus and thereby modulate cellular responses.

A Mammalian patched Homolog Is Expressed in Target Tissues of sonic hedgehog and Maps to a Region Associated with Developmental Abnormalities

Drosophila patched is a segment polarity gene required for the correct patterning of larval segments and imaginal discs during fly development and has a close functional relationship with hedgehog. We have isolated a complete human PATCHED cDNA sequence, which encodes a putative protein of 1296 amino acids, and displays 39% identity and 60% similarity to the Drosophila PATCHED protein. Hydropathy analysis suggests that human PATCHED is an integral membrane protein with a pattern of hydrophobic and hydrophilic stretches nearly identical to that of Drosophila patched. In the developing mouse embryo, patched is initially detected within the ventral neural tube and later in the somites and limb buds. Expression in the limb buds is restricted to the posterior ectoderm surrounding the zone of polarizing activity. The results show that patched is expressed in target tissues of sonic hedgehog, a murine homolog of Drosophila hedgehog suggesting that patched/hedgehog interactions have been conserved during evolution. Human PATCHED maps to human chromosome 9q22.3, the candidate region for the nevoid basal cell carcinoma syndrome. Patched expression is compatible with the congenital defects observed in the nevoid basal cell carcinoma syndrome.

Exon skipping and circular RNA formation in transcripts of the human cytochrome P-450 2C18 gene in epidermis and of the rat androgen binding protein gene in testis.

The cytochrome P-450 2C18 gene was found by reverse transcription-PCR to represent the most abundantly expressed gene of the P-450 2C subfamily in human epidermis. However, in addition to the canonical mRNA of nine exons, transcripts that have skipped exon 4 or 5, exons 4, 5, and 6, or exons 4, 5, 6, and 7 were also identified in this tissue. Remarkably, circular RNA transcripts synthesized by the joining of the donor and acceptor splice sites of the same exon were detected in human epidermis for exons 4 and 5. Moreover, molecules composed of exons 4, 5, and 6 with the donor splice site of exon 6 joined to the acceptor splice site of exon 4 or composed of exons 4, 5, 6, and 7 with the donor splice site of exon 7 joined to the acceptor splice site of exon 4 were also found to be present in this tissue. In rat testis, a similar analysis allowed the detection of a circular RNA molecule composed of exons 6 and 7 of the androgen binding protein (ABP) gene, with the donor splice site of exon 7 joined to the acceptor splice site of exon 6, and of an ABP mRNA which had skipped exons 6 and 7. These results apparently substantiate the hypothesis that alternative pre-mRNA splicing has the potential to generate not only mRNAs that lack one or more exons but also circular RNA molecules that are composed of the exons that are skipped. However, additional 2C18 circular species containing various combinations of exons were also detected in human epidermis, and an exon 6-skipped ABP mRNA molecule was identified in rat testis. This observation is interpreted as indicative that at low frequency, numerous circular RNA formation and exon skipping events may occur, allowing the joining of a variety of different combinations of exons. Moreover, the relative stability of these molecules is apparently the key factor that determines the relative ease of their detection.

Regulation of sexual differentiation in drug and steroid metabolism

Certain members of the cytochrome P-450 family are expressed at different levels in the livers of male and female rats. Although little is known of the functional significance of these sex differences, progress has been made towards the understanding of the endocrine control of hepatic sex differences in cytochrome P-450 levels. Jan-Ake Gustafsson and colleagues describe a subpopulation of hepatic sexually differentiated P-450s that is regulated by sex differences in growth hormone (GH) secretory pattern. This secretory pattern is in turn regulated by gonadal steroids. These studies demonstrate a novel action of GH and suggest that the hormonal secretory rhythm is pivotal in determination of biological effects.

Neighboring gene regulation by antisense long non-coding RNAs.

Antisense transcription, considered until recently as transcriptional noise, is a very common phenomenon in human and eukaryotic transcriptomes, operating in two ways based on whether the antisense RNA acts in cis or in trans. This process can generate long non-coding RNAs (lncRNAs), one of the most diverse classes of cellular transcripts, which have demonstrated multifunctional roles in fundamental biological processes, including embryonic pluripotency, differentiation and development. Antisense lncRNAs have been shown to control nearly every level of gene regulation—pretranscriptional, transcriptional and posttranscriptional—through DNA–RNA, RNA–RNA or protein–RNA interactions. This review is centered on functional studies of antisense lncRNA-mediated regulation of neighboring gene expression. Specifically, it addresses how these transcripts interact with other biological molecules, nucleic acids and proteins, to regulate gene expression through chromatin remodeling at the pretranscriptional level and modulation of transcriptional and post-transcriptional processes by altering the sense mRNA structure or the cellular compartmental distribution, either in the nucleus or the cytoplasm.

Alternative first exons of PTCH1 are differentially regulated in vivo and may confer different functions to the PTCH1 protein.

The PTCH1 gene is a human tumour suppressor gene frequently mutated in basal cell carcinoma (BCC) and several other tumour types. It encodes a receptor for soluble factors of the hedgehog family. Binding of hedgehog to the receptor relieves its inhibitory action on the transmembrane co-receptor Smoh. In this study we describe alternative first exons of the PTCH1 tumour suppressor gene and show that they are differentially regulated in normal tissues, exon 1B being expressed at very low levels and the major mRNA species containing exon 1 or 1A. Exon 1B transcripts were found to be specifically upregulated in nodular BCCs. The different PTCH1 transcripts all encode proteins that interact with Smoh in doubly transfected cells. Furthermore, functional assays demonstrated that whereas all PTCH1 isoforms can inhibit the activity of SHH, only the PTCH1B isoform is capable of fully inhibiting Smoh activity. The results indicate that in tumour cells the PTCH1B promoter is specifically activated and importantly, that the N-terminal part of PTCH1 including exon 1B is required for full inhibition of Smoh signaling but not for physical interaction with Smoh.

Distinct roles of PTCH2 splice variants in Hedgehog signalling.

The human PTCH2 gene is highly similar to PTCH1, a tumour suppressor gene frequently mutated in basal cell carcinoma and several other tumour types. PTCH1 is a transmembrane protein believed to inhibit another transmembrane protein SMO (Smoothened), which mediates HH (Hedgehog) signalling. In this study, we analysed the biological properties of several PTCH2 splice variants. An mRNA form that lacked the last exon was abundantly expressed in all tissues examined, in contrast with the one that included it. Moreover, a transcript lacking exon 9, which is a part of a conserved sterol-sensing domain, was identified in intestine, prostate and cerebellum. In ovary, spleen, testis, cerebellum and skin, an mRNA lacking both exons 9 and 10 could also be observed. The different PTCH2 isoforms localized in the cytoplasm were capable of internalizing the N-terminal fragment of Sonic HH (Shh-N). Additionally, the PTCH2 gene was found to be a target of HH signalling. PTCH2 promoter regulation assays demonstrated that only one of the PTCH2 variants could inhibit the activity of SHH-N, whereas none was capable of inhibiting the activated form of SMO (SMO-M2) and this contrasts with PTCH1. Despite the fact that the PTCH2 isoforms lacked the ability to inhibit SMO-M2 activity, all PTCH2 variants as well as PTCH1, on co-transfection with Smo, were able to change Smo localization from being largely dispersed in the cytoplasm to the juxtanuclear region. Furthermore, the PTCH2 isoforms and PTCH1 co-localized in doubly transfected cells and an interaction between them was confirmed using immunoprecipitation assays. Using Ptch1-/- mouse cells, it was shown that the PTCH2 variants and PTCH1 differentially act to reconstitute not only the SHH but also the Desert HH-dependent transcriptional response. We conclude that in spite of their structural similarities, the PTCH2 isoforms have distinct functional properties when compared with PTCH1.

MicroRNA-203 functions as a tumor suppressor in basal cell carcinoma

Basal cell carcinoma (BCC) of the skin represents the most common malignancy in humans. MicroRNAs (miRNAs), small regulatory RNAs with pleiotropic function, are commonly misregulated in cancer. Here we identify miR-203, a miRNA abundantly and preferentially expressed in skin, to be downregulated in BCCs. We show that activation of the Hedgehog (HH) pathway, critically involved in the pathogenesis of BCCs, as well as the EGFR/MEK/ERK/c-JUN signaling pathway suppresses miR-203. We identify c-JUN, a key effector of the HH pathway, as a novel direct target for miR-203 in vivo. Further supporting the role of miR-203 as a tumor suppressor, in vivo delivery of miR-203 mimics in a BCC mouse model results in the reduction of tumor growth. Our results identify a regulatory circuit involving miR-203 and c-JUN, which provides functional control over basal cell proliferation and differentiation. We propose that miR-203 functions as a ‘bona fide’ tumor suppressor in BCC, whose suppressed expression contributes to oncogenic transformation via derepression of multiple stemness- and proliferation-related genes, and its overexpression could be of therapeutic value.

Targeting the hedgehog signal transduction pathway at the level of GLI inhibits neuroblastoma cell growth in vitro and in vivo

Hedgehog (HH) signaling is an important regulator of embryogenesis that has been associated with the development of several types of cancer. HH signaling is characterized by Smoothened (SMO)‐dependent activation of the GLI transcription factors, which regulate the expression of critical developmental genes. Neuroblastoma, an embryonal tumor of the sympathetic nervous system, was recently shown to express high levels of key molecules in this signaling cascade. Using compounds blocking SMO (cyclopamine and SANT1) or GLI1/GLI2 (GANT61) activity revealed that inhibition of HH signaling at the level of GLI was most effective in reducing neuroblastoma growth. GANT61 sensitivity positively correlated to GLI1 and negatively to MYCN expression in the neuroblastoma cell lines tested. GANT61 downregulated GLI1, c‐MYC, MYCN and Cyclin D1 expression and induced apoptosis of neuroblastoma cells. The effects produced by GANT61 were mimicked by GLI knockdown but not by SMO knockdown. Furthermore, GANT61 enhanced the effects of chemotherapeutic drugs used in the treatment of neuroblastoma in an additive or synergistic manner and reduced the growth of established neuroblastoma xenografts in nude mice. Taken together this study suggests that inhibition of HH signaling is a highly relevant therapeutic target for high‐risk neuroblastoma lacking MYCN amplification and should be considered for clinical testing.