Kavita Y. Sarin

Conditional telomerase induction causes proliferation of hair follicle stem cells

TERT, the protein component of telomerase, serves to maintain telomere function through the de novo addition of telomere repeats to chromosome ends, and is reactivated in 90% of human cancers. In normal tissues, TERT is expressed in stem cells and in progenitor cells, but its role in these compartments is not fully understood. Here we show that conditional transgenic induction of TERT in mouse skin epithelium causes a rapid transition from telogen (the resting phase of the hair follicle cycle) to anagen (the active phase), thereby facilitating robust hair growth. TERT overexpression promotes this developmental transition by causing proliferation of quiescent, multipotent stem cells in the hair follicle bulge region. This new function for TERT does not require the telomerase RNA component, which encodes the template for telomere addition, and therefore operates through a mechanism independent of its activity in synthesizing telomere repeats. These data indicate that, in addition to its established role in extending telomeres, TERT can promote proliferation of resting stem cells through a non-canonical pathway.

TERT Promotes Epithelial Proliferation through Transcriptional Control of a Myc- and Wnt-Related Developmental Program

Telomerase serves a critical role in stem cell function and tissue homeostasis. This role depends on its ability to synthesize telomere repeats in a manner dependent on the reverse transcriptase (RT) function of its protein component telomerase RT (TERT), as well as on a novel pathway whose mechanism is poorly understood. Here, we use a TERT mutant lacking RT function (TERTci) to study the mechanism of TERT action in mammalian skin, an ideal tissue for studying progenitor cell biology. We show that TERTci retains the full activities of wild-type TERT in enhancing keratinocyte proliferation in skin and in activating resting hair follicle stem cells, which triggers initiation of a new hair follicle growth phase and promotes hair synthesis. To understand the nature of this RT-independent function for TERT, we studied the genome-wide transcriptional response to acute changes in TERT levels in mouse skin. We find that TERT facilitates activation of progenitor cells in the skin and hair follicle by triggering a rapid change in gene expression that significantly overlaps the program controlling natural hair follicle cycling in wild-type mice. Statistical comparisons to other microarray gene sets using pattern-matching algorithms revealed that the TERT transcriptional response strongly resembles those mediated by Myc and Wnt, two proteins intimately associated with stem cell function and cancer. These data show that TERT controls tissue progenitor cells via transcriptional regulation of a developmental program converging on the Myc and Wnt pathways.

Genomic Analysis of Smoothened Inhibitor Resistance in Basal Cell Carcinoma

Smoothened (SMO) inhibitors are under clinical investigation for the treatment of several cancers. Vismodegib is approved for the treatment of locally advanced and metastatic basal cell carcinoma (BCC). Most BCC patients experience significant clinical benefit on vismodegib, but some develop resistance. Genomic analysis of tumor biopsies revealed that vismodegib resistance is associated with Hedgehog (Hh) pathway reactivation, predominantly through mutation of the drug target SMO and to a lesser extent through concurrent copy number changes in SUFU and GLI2. SMO mutations either directly impaired drug binding or activated SMO to varying levels. Furthermore, we found evidence for intra-tumor heterogeneity, suggesting that a combination of therapies targeting components at multiple levels of the Hh pathway is required to overcome resistance.

Reversible cell-cycle entry in adult kidney podocytes through regulated control of telomerase and Wnt signaling

Mechanisms of epithelial cell renewal remain poorly understood in the mammalian kidney, particularly in the glomerulus, a site of cellular damage in chronic kidney disease. Within the glomerulus, podocytes—differentiated epithelial cells crucial for filtration—are thought to lack substantial capacity for regeneration. Here we show that podocytes rapidly lose differentiation markers and enter the cell cycle in adult mice in which the telomerase protein component TERT is conditionally expressed. Transgenic TERT expression in mice induces marked upregulation of Wnt signaling and disrupts glomerular structure, resulting in a collapsing glomerulopathy resembling those in human disease, including HIV-associated nephropathy (HIVAN). Human and mouse HIVAN kidneys show increased expression of TERT and activation of Wnt signaling, indicating that these are general features of collapsing glomerulopathies. Silencing transgenic TERT expression or inhibiting Wnt signaling through systemic expression of the Wnt inhibitor Dkk1 in either TERT transgenic mice or in a mouse model of HIVAN results in marked normalization of podocytes, including rapid cell-cycle exit, re-expression of differentiation markers and improved filtration barrier function. These data reveal an unexpected capacity of podocytes to reversibly enter the cell cycle, suggest that podocyte renewal may contribute to glomerular homeostasis and implicate the telomerase and Wnt–β-catenin pathways in podocyte proliferation and disease.

Aging, graying and loss of melanocyte stem cells.

Hair graying is one of the prototypical signs of human aging. Maintenance of hair pigmentation is dependent on the presence and functionality of melanocytes, neural crest derived cells which synthesize pigment for growing hair. The melanocytes, themselves, are maintained by a small number of stem cells which reside in the bulge region of the hair follicle. The recent characterization of the melanocyte lineage during aging has significantly accelerated our understanding of how age-related changes in the melanocyte stem cell compartment contribute to hair graying. This review will discuss our current understanding of hair graying, drawing on evidence from human and mouse studies, and consider the contribution of melanocyte stem cells to this process. Furthermore, using the melanocyte lineage as an example, it will discuss common theories of tissue and stem cell aging.

Mosaic Activating RAS Mutations in Nevus Sebaceus and Nevus Sebaceus Syndrome

To the editor: Nevus sebaceus is a common congenital skin hamartoma, classically appearing as a yellow-hued plaque on the scalp, face, or neck. It is the hallmark lesion of Schimmelpenning/nevus sebaceus syndrome (MIM: 163200), a multisystem disorder that includes a spectrum of central nervous system, ocular, skeletal, and cardiovascular defects. Secondary neoplasms arise within nevus sebaceus at a modest but elevated rate (Moody et al, 2012), prompting disagreement about whether they should be routinely excised (Shwayder, 2011). Determining the pathogenesis of nevus sebaceus would provide a framework to better understand this lesion and its associated syndrome. The appearance of nevus sebaceus along Blaschko’s lines suggests that a mosaic genetic mutation causes the lesion, with more extensive multisystem involvement potentially underlying the syndromic form (Happle, 1993). Here, we report a case of an individual with nevus sebaceus syndrome. As individuals with this syndrome are uncommon, we sought to identify associated mutations by comparing the exome sequence of the nevus sebaceus from our patient with those of sporadic nevus sebaceus. Our index patient is a 38-year old female who was born with Chiari malformation, myelomeningocele, and resultant paraplegia. Due to hydrocephalus and marked ventricomegaly, she required ventriculo-peritoneal shunt placement. Imaging studies demonstrated rotoscoliosis. She has had cognitive developmental delay and suffered from generalized seizures during childhood. In her early thirties, she experienced a middle right cerebral artery stroke. Despite her condition, she remains high functioning and lives in an assisted care facility. No other family members are affected by similar medical conditions, and no cause has been attributed to her findings. In the past year, the patient became bothered by growths on her forehead and presented to our clinic. On examination, she exhibited frontal bossing and a ~4 cm × ~3 cm yellow-tan, papillomatous plaque on the paramidline forehead that had been present since birth (Figure 1a). Several pedunculated papules were noted within the lesion. No other significant cutaneous findings were appreciated. Figure 1 Clinical and histologic features of a patient with nevus sebaceus syndrome Per patient request, the lesion was excised and a portion of the excision specimen was collected with her written informed consent. Our study complied with the Declaration of Helsinki Principles and was approved by the Stanford IRB. Histologic evaluation confirmed features of nevus sebaceus with no secondary neoplasms (Figure 1b). Accordingly, in light of the extensive neurological and skeletal involvement, a diagnosis of Schimmelpenning/nevus sebaceus syndrome was made. In efforts to determine an underlying genetic mutation, four additional, independent nevus sebaceus were collected from elective excisions along with adjacent normal skin controls. The five samples were subjected to exome sequencing and analyzed for mutations using Seqgene (Deng, 2011) and DNAnexus (www.dnanexus.com) as described in the Supplemental Text. Analysis of recurrent lesion-specific variants identified an HRAS point mutation (c.37G>C, p.Gly13Arg) in the index case and 2 of 4 isolated nevus sebaceus, with a variant allele frequency ranging from 17–43%. Sanger sequencing confirmed the HRAS mutation in all five lesional samples and its absence in all matched controls (Figure 2a). Examination of the two HRAS mutation-negative exomes showed low sequence coverage (<20 reads) at the mutation site, which may account for the false-negative calls. Figure 2 Activating mosaic RAS mutations in nevus sebaceus Lesions arising along Blaschko’s lines are hypothesized to stem from a mosaic mutation affecting a specific cell lineage during development. To evaluate whether the candidate mutation fit this criterion, we used laser capture microdissection to isolate DNA from the lesional epidermis and dermis from the index case and one of the sporadic nevus sebaceus. In both cases, the mutation was limited to the epidermis, supporting the hypothesis of an acquired mutation affecting ectodermal precursors (Figure 2b). Both alleles were represented in approximately equal intensities, indicating that the mutation is likely heterozygous. We next performed targeted Sanger sequencing on a validation set of 31 independent nevus sebaceus from archived tissues, and identified the HRAS p.Gly13Arg mutation in 24/31 samples and p.Gly12Asp in one sample. Remaining mutation-negative cases were evaluated for KRAS and NRAS hotspot mutations, identifying two samples carrying KRAS p.Gly12Asp mutations. Six validation samples had patient-matched normal skin tissue available, and the corresponding RAS mutations were absent in all six control samples. In total, 32 of 36 samples (89%) demonstrated HRAS or KRAS mutations, confirming a strong correlation between activating RAS mutations and nevus sebaceus (Figure 2c). We suspect that the remaining negative cases may be due to genetic heterogeneity, or to a low mutant allele frequency secondary to admixture with normal tissue. RAS promotes cell growth through activation of multiple pathways, chief among them the mitogen-activated protein kinase (MAPK) signal transduction pathway. Activating mutations in this gene family have well-established links to cancer (Schubbert et al, 2007). Germline activating HRAS mutations cause Costello syndrome, which features predisposition to neoplasia and development of cutaneous papillomas (Gripp and Lin, 2012). Taken together, the known biologic features of activated RAS genes are consistent with the hamartomatous overgrowth and elevated neoplasia risk observed in nevus sebaceus. To evaluate RAS-MAPK signaling, we performed phosphorylated ERK (pERK) staining on a set of nevi with confirmed HRAS mutations. Immunohistochemistry revealed increased pERK staining in lesional vs. normal epidermis, consistent with RAS-MAPK hyperactivation (Figure 2d). In one sample, a squamous cell carcinoma was identified arising from nevus sebaceus, highlighted by elevated p16 staining (Hodges and Smoller, 2002). The pattern of neoplasia arising from a background of upregulated pERK supports the hypothesis that RAS-MAPK hyperactivation may predispose towards development of secondary neoplasms in nevus sebaceus. Basal cell carcinomas were once thought to arise commonly from nevus sebaceus, but others have subsequently contended that the majority of these tumors are actually trichoblastomas (Cribier et al, 2000). Our data provide genetic support for the latter opinion, as most basal cell carcinomas arise from Hedgehog pathway dysregulation and lack RAS mutations (Reifenberger et al, 2005). Our findings also raise the possibility that tumors arising from nevus sebaceus, such as syringocystadenoma papilliferum and trichoblastomas, may be associated with RAS mutations as well. Using targeted sequencing and SNaPshot assays, Hafner and colleagues have recently profiled oncogenic hotspot mutations in epidermal and sebaceous nevi (Groesser et al, 2012; Hafner et al, 2012). Together with the results presented here and by others in this issue (Levinsohn et al, 2012), the cumulative data demonstrate that keratinocytic epidermal nevi and sebaceous nevi are both associated with activating HRAS p.Gly13Arg and KRAS p.Gly12Asp mutations, supporting the belief held by some clinicians they represent a spectrum of the same entity (Sybert, 2010). We postulate that the phenotypic difference between these nevi may be related to the extent of the mutation, as well as body site-specific embryologic patterns and environment. The knowledge of the genetic basis of nevus sebaceus and its associated syndrome represents a further step towards understanding genotype-phenotype correlations arising from genetic mosaicism.

An Investigator-Initiated Open-Label Trial of Sonidegib in Advanced Basal Cell Carcinoma Patients Resistant to Vismodegib

Purpose: To assess the tumor response to the smoothened (SMO) inhibitor, sonidegib (LDE225), in patients with an advanced basal cell carcinoma (BCC) resistant to treatment with vismodegib (GDC0449). Experimental Design: Nine patients with an advanced BCC that was previously resistant to treatment with vismodegib were given sonidegib in this investigational, open-label study. Tumor response was determined using the response evaluation criteria in solid tumors. SMO mutations were identified using biopsy samples from the target BCC location. Results: The median duration of treatment with sonidegib was 6 weeks (range, 3–58 weeks). Five patients experienced progressive disease with sonidegib. Three patients experienced stable disease and discontinued sonidegib either due to adverse events (n = 1) or due to election for surgery (n = 2). The response of one patient was not evaluable. SMO mutations with in vitro data suggesting resistance to Hh pathway inhibition were identified in 5 patients, and none of these patients experienced responses while on sonidegib. Conclusions: Patients with advanced BCCs that were previously resistant to treatment with vismodegib similarly demonstrated treatment resistance with sonidegib. Patients who have developed treatment resistance to an SMO inhibitor may continue to experience tumor progression in response to other SMO inhibitors. Clin Cancer Res; 22(6); 1325–9. ©2015 AACR.

Effects of Combined Treatment With Arsenic Trioxide and Itraconazole in Patients With Refractory Metastatic Basal Cell Carcinoma

IMPORTANCE Tumor resistance is an emerging problem for Smoothened (SMO) inhibitor-treated metastatic basal cell carcinoma (BCC). Arsenic trioxide and itraconazole antagonize the hedgehog (HH) pathway at sites distinct from those treated by SMO inhibitors. OBJECTIVE To determine whether administration of intravenous arsenic trioxide and oral itraconazole in patients with metastatic BCC is associated with a reduction in GLI1 messenger RNA expression in tumor and/or normal skin biopsy samples. DESIGN, SETTING, AND PARTICIPANTS Five men with metastatic BCC who experienced relapse after SMO inhibitor treatment underwent intravenous arsenic trioxide treatment for 5 days, every 28 days, and oral itraconazole treatment on days 6 to 28. Data were collected from April 10 to November 14, 2013. Follow-up was completed on October 3, 2015, and data were analyzed from June 5 to October 6, 2015. MAIN OUTCOMES AND MEASURES The primary outcome was the change in messenger RNA levels of the GLI family zinc finger 1 (GLI1) gene (HH-pathway target gene) in biopsy specimens of normal skin or BCC before and after treatment. Secondary objectives were evaluation of tumor response and tolerability. RESULTS Of the 5 patients (mean [SD] age, 52 [9] years; age range, 43-62 years), 3 completed 3 cycles of treatment and 2 discontinued treatment early owing to disease progression or adverse events. Adverse effects included grade 2 transaminitis and grade 4 leukopenia with a grade 3 infection. Overall, arsenic trioxide and itraconazole reduced GLI1 messenger RNA levels by 75% from baseline (P < .001). The best overall response after 3 treatment cycles was stable disease in 3 patients. CONCLUSIONS AND RELEVANCE Targeting the HH pathway with sequential arsenic trioxide and itraconazole treatment is a feasible treatment for metastatic BCC. Although some patients experienced stable disease for 3 months, none had tumor shrinkage, which may be owing to transient GLI1 suppression with sequential dosing. Continuous dosing may be required to fully inhibit the HH pathway and achieve clinical response.

Squamous Change in Basal-Cell Carcinoma with Drug Resistance

In a 62-year-old woman with advanced basal-cell carcinoma who was treated with vismodegib, a recurrent mass was identified as a squamous-cell carcinoma that shared genetic features with the primary basal-cell tumor.

Familial skin cancer syndromes: Increased melanoma risk

Phenotypic traits, such as red hair and freckling, increase melanoma risk by 2- to 3-fold. In addition, approximately 10% of melanomas are caused by inherited germline mutations that increase melanoma risk from 4- to >1000-fold. This review highlights the key genes responsible for inherited melanoma, with an emphasis on when a patient should undergo genetic testing. Many genetic syndromes associated with increased melanoma risk are also associated with an increased risk of other cancers. Identification of these high-risk patients is essential for preventive behavior reinforcement, genetic counseling, and ensuring other required cancer screenings.