Rune Toftgård

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.

Lgr5 marks cycling, yet long-lived, hair follicle stem cells

In mouse hair follicles, a group of quiescent cells in the bulge is believed to have stem cell activity. Lgr5, a marker of intestinal stem cells, is expressed in actively cycling cells in the bulge and secondary germ of telogen hair follicles and in the lower outer root sheath of anagen hair follicles. Here we show that Lgr5+ cells comprise an actively proliferating and multipotent stem cell population able to give rise to new hair follicles and maintain all cell lineages of the hair follicle over long periods of time. Lgr5+ progeny repopulate other stem cell compartments in the hair follicle, supporting the existence of a stem or progenitor cell hierarchy. By marking Lgr5+ cells during trafficking through the lower outer root sheath, we show that these cells retain stem cell properties and contribute to hair follicle growth during the next anagen. Expression analysis suggests involvement of autocrine Hedgehog signaling in maintaining the Lgr5+ stem cell population.

Inhibition of GLI-mediated transcription and tumor cell growth by small-molecule antagonists.

The developmentally important Hedgehog (Hh) signaling pathway has recently been implicated in several forms of solid cancer. Current drug development programs focus on targeting the protooncogene Smoothened, a key transmembrane pathway member. These drug candidates, albeit promising, do not address the scenario in which pathway activation occurs downstream of Smoothened, as observed in cases of medulloblastoma, glioma, pericytoma, breast cancer, and prostate cancer. A cellular screen for small-molecule antagonists of GLI-mediated transcription, which constitutes the final step in the Hh pathway, revealed two molecules that are able to selectively inhibit GLI-mediated gene transactivation. We provide genetic evidence of downstream pathway blockade by these compounds and demonstrate the ineffectiveness of upstream antagonists such as cyclopamine in such situations. Mechanistically, both inhibitors act in the nucleus to block GLI function, and one of them interferes with GLI1 DNA binding in living cells. Importantly, the discovered compounds efficiently inhibited in vitro tumor cell proliferation in a GLI-dependent manner and successfully blocked cell growth in an in vivo xenograft model using human prostate cancer cells harboring downstream activation of the Hh pathway.

Lgr6 marks stem cells in the hair follicle that generate all cell lineages of the skin.

Hair Today, Skin Tomorrow The epidermis of mammals contains hair follicles, sebaceous glands, and interfollicular epidermis, but it has not been clear how the development and repair of these structures is regulated. Snippert et al. (p. 1385) show that a stem-cell cluster in the hair follicle, characterized by the expression of Lgr6, a close homolog of the Lgr5 marker for stem cells in the small intestine and colon, resides directly above the hair bulge and gives rise to all cell lineages of the skin. Skin wounds in adult mice are repaired by Lgr6 stem cells in the hair follicles that flank the damage. After hair morphogenesis, Lgr6 stem cells give rise to epidermal and sebaceous gland lineages to generate fully differentiated new skin. Skin wounds can be repaired by primitive stem cells into fully differentiated tissue, complete with hairs and sebaceous glands. Mammalian epidermis consists of three self-renewing compartments: the hair follicle, the sebaceous gland, and the interfollicular epidermis. We generated knock-in alleles of murine Lgr6, a close relative of the Lgr5 stem cell gene. Lgr6 was expressed in the earliest embryonic hair placodes. In adult hair follicles, Lgr6+ cells resided in a previously uncharacterized region directly above the follicle bulge. They expressed none of the known bulge stem cell markers. Prenatal Lgr6+ cells established the hair follicle, sebaceous gland, and interfollicular epidermis. Postnatally, Lgr6+ cells generated sebaceous gland and interfollicular epidermis, whereas contribution to hair lineages gradually diminished with age. Adult Lgr6+ cells executed long-term wound repair, including the formation of new hair follicles. We conclude that Lgr6 marks the most primitive epidermal stem cell.

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.

Hedgehog beyond medulloblastoma and basal cell carcinoma

The Hedgehog (Hh) signaling pathway is of central importance during embryo development in metazoans and governs a diverse array of processes including cell proliferation, differentiation, and tissue patterning. In normal adult physiology, the pathway is implicated in stem cell maintenance, tissue repair and regeneration. However, the pathways darker side is its involvement in several types of human cancer, to which it confers growth promoting and/or survival capabilities to the cancer cell to varying degrees, and by different mechanisms. The Hh pathway is firmly linked to the etiology of basal cell carcinoma and to at least a subset of medulloblastoma. There is increasing evidence that other sporadic cancers, including those in pancreas, prostate, lung, and breast, could also be dependent on Hh pathway activity. In this review, we provide an overview of the pathways role in various tumor types, where much of the framework for Hh-dependent malignancies has been elucidated in experimental mouse models. We discuss three different signal transduction models for the pathways involvement in cancer: i) ligand-independent signaling, ii) ligand-dependent autocrine/juxtacrine signaling, and iii) ligand-dependent paracrine signaling. These different modes of signaling may have implications for future therapeutic interventions aimed at inhibiting the pathway during disease. In addition, crosstalk with other pathways, and indications of non-canonical Hh signaling in cancer cells may further cause complications, or perhaps possibilities, in the treatment regimen. Finally, we review the rapid progress and promising results in the development of small-molecule inhibitors of the Hh pathway.

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.

Indian hedgehog is a major mediator of progesterone signaling in the mouse uterus

The hedgehog family of morphogens are regulators of cell proliferation, differentiation and cell-cell communication. These morphogens have been shown to have important roles in organogenesis, spermatogenesis, stem cell maintenance and oncogenesis. Indian hedgehog (encoded by Ihh) has been shown to be expressed in the uterine epithelium under the control of the steroid hormone, progesterone. Although in vivo and in vitro studies have shown that progesterone achieves its effects on uterine function through epithelial-stromal cross-talk, molecular mediator(s) for this cellular communication pathway have not been elucidated. Using new experimental approaches that ablate Ihh specifically in Pgr-positive uterine cells of the mouse, we demonstrate that Ihh is an essential mediator of Pgr action in the uterus, and expression of this factor is critical in mediating the communication between the uterine epithelium and stroma required for embryo implantation.

Deregulation of the hedgehog signalling pathway: a possible role for the PTCH and SUFU genes in human rhabdomyoma and rhabdomyosarcoma development.

The naevoid basal cell carcinoma syndrome (NBCCS) is caused by mutations in the hedgehog receptor PTCH gene. It is characterized by developmental defects and a predisposition to the development of certain tumours, such as basal cell carcinoma, medulloblastoma and meningioma, and potentially fetal rhabdomyomas and embryonal rhabdomyosarcomas. This study aimed to analyse PTCH status in an NBCCS patient with fetal rhabdomyoma and to investigate whether deregulation of hedgehog signalling, as shown by altered expression of hedgehog pathway components and/or genetic imbalances, is a general finding in sporadic rhabdomyomas and rhabdomyosarcomas. The NBCCS patient had a novel PTCH germ‐line mutation, 1370insT, and developed a fetal rhabdomyoma that harboured a 30 bp in‐frame deletion in the second allele resulting in homozygous inactivation of PTCH. Sporadic rhabdomyomas and rhabdomyosarcomas showed overexpression of PTCH (43/43) and GLI1 (41/43) mRNA, as determined by in situ hybridization, indicating ongoing active hedgehog signalling. Immunohistochemical staining revealed a subgroup of fetal rhabdomyomas and embryonal rhabdomyosarcomas (12/34) lacking PTCH immunoreactivity. Four of nine informative fetal rhabdomyomas and embryonal rhabdomyosarcomas showed loss of heterozygosity (LOH) in the PTCH region with two of these (one fetal rhabdomyoma and one embryonal rhabdomyosarcoma) also showing LOH in the SUFU region. These findings suggest that haploinsufficiency for the two tumour suppressor genes PTCH and SUFU, which are both active in the same signalling pathway, may be important for tumour development. Based on our results we propose that the pathogenesis of rhabdomyoblastic tumours, particularly fetal rhabdomyomas and embryonal rhabdomyosarcomas, involves deregulation of the hedgehog signalling pathway. Copyright

Non-Canonical Activation of GLI Transcription Factors: Implications for Targeted Anti-Cancer Therapy

GLI transcription factors constitute the final effectors of the Hedgehog (HH) signaling pathway. In many tumors, such as those of the pancreas, prostate, skin or lung, ectopic activation of GLI proteins has been linked to tumorigenesis. In several of these cases, HH ligand- or receptor-induced signaling (canonical HH signaling) was found to be the cause underlying GLI activation. Recent evidence points towards additional, non-canonical, mechanisms of GLI activation. Here we review findings on the crosstalk of two HH-unrelated signaling pathways (RAS and Transforming growth factor β) to the HH pathway downstream of the signaling component Smoothened. Both pathways stimulate and/or induce GLI1 and GLI2 activity independent of the presence of HH ligands. We also discuss the putative roles of these crosstalk mechanisms for tumor cell metastasis. The emerging picture of GLI transcription factors as an integrative platform of numerous signaling inputs has important implications for the understanding of tumor development and argues for inclusion of targets acting downstream of the receptor level in the design of current drug development programs.