Ji-an Wang

Sun exposure causes somatic second hit mutations and angiofibroma development in Tuberous Sclerosis Complex

Tuberous sclerosis complex (TSC) is characterized by the formation of tumors in multiple organs and is caused by germline mutation in one of two tumor suppressor genes, TSC1 and TSC2. As for other tumor suppressor gene syndromes, the mechanism of somatic second-hit events in TSC tumors is unknown. We grew fibroblast-like cells from 29 TSC skin tumors from 22 TSC subjects and identified germline and second-hit mutations in TSC1/TSC2 using next-generation sequencing. Eighteen of 22 (82%) subjects had a mutation identified, and 8 of the 18 (44%) subjects were mosaic with mutant allele frequencies of 0 to 19% in normal tissue DNA. Multiple tumors were available from four patients, and in each case, second-hit mutations in TSC2 were distinct indicating they arose independently. Most remarkably, 7 (50%) of the 14 somatic point mutations were CC>TT ultraviolet signature mutations, never seen as a TSC germline mutation. These occurred exclusively in facial angiofibroma tumors from sun-exposed sites. These results implicate UV-induced DNA damage as a cause of second-hit mutations and development of TSC facial angiofibromas and suggest that measures to limit UV exposure in TSC children and adults should reduce the frequency and severity of these lesions.

TSC2 Loss in Lymphangioleiomyomatosis Cells Correlated with Expression of CD44v6, a Molecular Determinant of Metastasis

Lymphangioleiomyomatosis (LAM), a rare multisystem disease found primarily in women of childbearing age, is characterized by the proliferation of abnormal smooth muscle-like cells, LAM cells, that form nodules in the pulmonary interstitium. Proliferation of LAM cells results, in part, from dysfunction in tuberous sclerosis complex (TSC) genes TSC1 (hamartin) and/or TSC2 (tuberin). Identification of LAM cells in donor lungs, their isolation from blood, and their presence in urine, chylous ascites, and pleural effusions are consistent with their ability to metastasize. Here, we investigated the presence on LAM cells of the hyaluronic acid receptor CD44 and its splice variants associated with metastasis. The heterogeneous populations of cells grown from lungs of 12 LAM patients contain cells expressing mRNA for the variant CD44v6. Histologically, CD44v6 was present in LAM lung nodules, but not in normal vascular smooth muscle cells. CD44v6-positive sorted cells showed loss of heterozygosity at the TSC2 locus; binding of CD44v6 antibody resulted in loss of cell viability. Levels of CD44 were higher in cultured Eker rat (Tsc2-/-) cells than in Tsc2+/+ cells, but unlike human LAM cells, the Tsc2-/- Eker rat cells did not contain CD44v6 splice variant mRNA. CD44 splicing and signaling is regulated by osteopontin. Plasma from LAM patients contained higher concentrations of osteopontin than plasma of healthy, age-, and sex-matched volunteers (P = 0.00003) and may be a biomarker for LAM. The cell surface receptor CD44 and its splice variant CD44v6 may contribute to the metastatic potential of LAM cells.

MCP-1 overexpressed in tuberous sclerosis lesions acts as a paracrine factor for tumor development.

Patients with tuberous sclerosis complex (TSC) develop hamartomatous tumors showing loss of function of the tumor suppressor TSC1 (hamartin) or TSC2 (tuberin) and increased angiogenesis, fibrosis, and abundant mononuclear phagocytes. To identify soluble factors with potential roles in TSC tumorigenesis, we screened TSC skin tumor–derived cells for altered gene and protein expression. Fibroblast-like cells from 10 angiofibromas and five periungual fibromas produced higher levels of monocyte chemoattractant protein-1 (MCP-1) mRNA and protein than did fibroblasts from the same patients normal skin. Conditioned medium from angiofibroma cells stimulated chemotaxis of a human monocytic cell line to a greater extent than conditioned medium from TSC fibroblasts, an effect blocked by neutralizing MCP-1–specific antibody. Overexpression of MCP-1 seems to be caused by loss of tuberin function because Eker rat embryonic fibroblasts null for Tsc2 (EEF Tsc2 −/−) produced 28 times as much MCP-1 protein as did EEF Tsc2 +/+ cells; transient expression of WT but not mutant human TSC2 by EEF Tsc2 −/− cells inhibited MCP-1 production; and pharmacological inhibition of the Rheb-mTOR pathway, which is hyperactivated after loss of TSC2, decreased MCP-1 production by EEF Tsc2 −/− cells. Together these findings suggest that MCP-1 is an important paracrine factor for TSC tumorigenesis and may be a new therapeutic target.

Mesenchymal–epithelial interactions involving epiregulin in tuberous sclerosis complex hamartomas

Patients with tuberous sclerosis complex (TSC) develop hamartomas containing biallelic inactivating mutations in either TSC1 or TSC2, resulting in mammalian target of rapamycin (mTOR) activation. Hamartomas overgrow epithelial and mesenchymal cells in TSC skin. The pathogenetic mechanisms for these changes had not been investigated, and the existence or location of cells with biallelic mutations (“two-hit” cells) was unclear. We compared TSC skin hamartomas (angiofibromas and periungual fibromas) with normal-appearing skin of the same patient, and we observed more proliferation and mTOR activation in hamartoma epidermis. Two-hit cells were not detected in the epidermis. Fibroblast-like cells in the dermis, however, exhibited allelic deletion of TSC2, in both touch preparations of fresh tumor samples and cells grown from TSC skin tumors, suggesting that increased epidermal proliferation and mTOR activation were not caused by second-hit mutations in the keratinocytes but by mesenchymal–epithelial interactions. Gene expression arrays, used to identify potential paracrine factors released by mesenchymal cells, revealed more epiregulin mRNA in fibroblast-like angiofibroma and periungual fibroma cells than in fibroblasts from normal-appearing skin of the same patient. Elevation of epiregulin mRNA was confirmed with real-time PCR, and increased amounts of epiregulin protein were demonstrated with immunoprecipitation. Epiregulin stimulated keratinocyte proliferation and phosphorylation of ribosomal protein S6 in vitro. These results suggest that hamartomatous TSC skin tumors are induced by paracrine factors released by two-hit cells in the dermis and that proliferation with mTOR activation of the overlying epidermis is an effect of epiregulin.

Human TSC2-null fibroblast-like cells induce hair follicle neogenesis and hamartoma morphogenesis

Hamartomas are composed of cells native to an organ but abnormal in number, arrangement or maturity. In the tuberous sclerosis complex (TSC), hamartomas develop in multiple organs because of mutations in TSC1 or TSC2. Here we show that TSC2-null fibroblast-like cells grown from human TSC skin hamartomas induced normal human keratinocytes to form hair follicles and stimulated hamartomatous changes. Follicles were complete with sebaceous glands, hair shafts and inner and outer root sheaths. TSC2-null cells surrounding the hair bulb expressed markers of the dermal sheath and dermal papilla. Tumour xenografts recapitulated characteristics of TSC skin hamartomas with increased mammalian target of the rapamycin complex 1 (mTORC1) activity, angiogenesis, mononuclear phagocytes and epidermal proliferation. Treatment with an mTORC1 inhibitor normalized these parameters and reduced the number of tumour cells. These studies indicate that TSC2-null cells are the inciting cells for TSC skin hamartomas, and suggest that studies on hamartomas will provide insights into tissue morphogenesis and regeneration.

Dissociated Human Dermal Papilla Cells Induce Hair Follicle Neogenesis in Grafted Dermal–Epidermal Composites

Tissue engineered skin substitutes are used in the clinic to treat chronic wounds and burns, and in the laboratory to advance our understanding of wound healing, skin biology, and skin disease. One type of skin substitute, dermal-epidermal composites (DEC), also known as skin equivalents or bilayered living skin constructs, are comprised of dermal fibroblasts embedded in a matrix such as collagen and overlaid with keratinocytes (Veves et al, 2001). DECs promote wound healing (Falanga and Sabolinski, 1999) and have been used to model skin development and diseases (Carretero et al., 2011, Kamsteeg et al., 2011), but their use has been limited by the inability of the skin constructs to regenerate hair follicles (HFs). n nDuring embryogenesis, mesenchymal cells signal the overlying epithelium to induce HF formation, and in adults a specialized group of mesenchymal cells, the dermal papilla (DP) cells, have been shown to retain the capacity to induce HF regeneration (Hardy 1992, Reddy et al., 2001, Gharzi et al., 2003). DP cells from rodents induce HFs in a variety of assays (reviewed in Ohyama et al., 2010), but it has been difficult to grow human DP cells that maintain inductive capacity in culture (Higgins et al., 2010). Recent technological advances have enabled the use of human cells to form chimeric HFs, for example by combining human keratinocytes and rodent mesenchymal cells in chamber assays (Ehama et al. 2007), human scalp dermal papilla cells and mouse epidermal keratinocytes in flap grafts (Qiao et al., 2009) or injecting human DP cells, grown as spheroids, together with mouse epidermal cells in reconstitution or “patch” assays (Kang et al., 2012). However, to date, complete and entirely human HFs formed from normal cultured cells have not been reported. Recently, the potential for human hair follicle development in grafted DECs was demonstrated using composites containing human neonatal foreskin keratinocytes (NFK) and fibroblast-like cells derived from tuberous sclerosis skin hamartomas (Li et al., 2011). Therefore, we used the conditions developed in these experiments to test for HF formation in DECs using normal human DP cells. n nHuman DP cells isolated from temporal scalp dermis (Promocell, Heidelberg, Germany) from six donors were propagated in vitro according to manufacturers recommendations. Alkaline phosphatase activity, a DP marker which correlates with hair-inducing capacity (Ohyama et al., 2010), was measured in vitro using the BCIP/NBT substrate (Sigma-Aldrich, St. Louis, MO) on passage 5 DP cells. Alkaline phosphatase activity was variable between samples, with cells from 3 of the donors showing alkaline phosphatase activity in more than 50% of the cells (Table 1). DECs were constructed by combining DP cells with rat tail collagen type 1, adding NFKs on top and bringing the constructs to the air-liquid interface for 2 days before grafting onto female nude mice. Eight weeks after grafting, HFs were observed in mice grafted with the 3 human DP cells with higher alkaline phosphatase activity (Table 1, Figure 1a). HFs had a bulb, dermal sheath, hair matrix and cortex (Figure 1b). Epithelial compartments of the HFs were intact with concentric layers of inner and outer root sheaths, sebaceous glands and hair shaft (Figure 1c-e). Fluorescence in situ hybridization showed the hybridization of a human-specific Alu probe (green) to the nuclei of both epithelial and dermal cells within the graft, including dermal sheath and dermal papilla, confirming their human origin (Figure 1f and g). An antibody reactive with human but not mouse COX IV stained follicular epithelium and dermal papilla/dermal sheath of grafts (Figure 1h). Fluorescence in situ hybridization showed the hybridization of a human-specific, pan-centromeric probe (green) to the nuclei of both epithelial and dermal components (Figure 1i), whereas a human-specific Y-chromosome probe (red) hybridized to nuclei in the epidermis and the follicular epithelium (Figure 1j), consistent with the origin of dermal and epidermal cells from female and male donors, respectively. HFs also stained for markers of specific compartments of a fully developed human HF. Cells in the region of the DP and lower DS displayed alkaline phosphatase activity (Figure 1k), normal reactivity with specific antibodies to human nestin (Figure 1l) and versican (Figure 1m). As expected, anagen HFs had more concentrated immunoreactivity to Ki-67 in the region of the hair matrix relative to the overlying epidermis (Figure 1n). The companion layer as identified by keratin 75 staining was present between the inner and outer root sheaths (Figure 1o). The basal layer of the outer root sheath was immunoreactive for keratin 15, a marker of HF stem cells located in the bulge region (Figure 1p). n n n nFigure 1 n nDe novo formation of human hair follicles in grafted dermal-epidermal constructs. Representative H&E stained sections of grafts showing (a) pilosebaceous units, (b) DP and matrix, (c, d) hair shaft, inner and outer root sheath, (d) sebaceous gland, ... n n n n n nTable 1 n nHuman dermal papilla cells with higher alkaline phosphatase activity form hair follicles when combined with neonatal foreskin keratinocytes in grafted dermal-epidermal constructs. n n n nIn summary, we report that cultured specialized human cells such as DP cells can induce complete pilosebaceous units in vivo in the grafted DEC model. Human HF formation may have been enabled by particular features of our experimental methods, such as the use of DP cells from the temporal scalp, use of an occlusive dressing for a long period after grafting, and a long duration for maturation of the grafts. It is not yet known whether these or other factors are critical to enable human HF formation in DECs, but success using the conditions described appears to require a starting population of DP cells in which the majority show alkaline phosphatase activity. This model could be used to evaluate the trichogenicity of various types of dermal cells in combination with different keratinocyte populations, evaluate hair loss therapies and may be adaptable to examine regeneration of other skin appendages and the formation of skin adnexal neoplasms. Next-generation skin substitutes that promote hair follicle neogenesis are expected to promote healing, normal skin function and appearance, and can be used to study of human HF neogenesis and regeneration with cultured adult cells.

Cytokeratin 15 expression in central, centrifugal, cicatricial alopecia: new observations in normal and diseased hair follicles

Background: Cytokeratin 15 (CK15) is a useful marker for the bulge zone (BZ) and has been used to examine follicles in cicatricial alopecia. We studied the expression of CK15 in hair follicles of patients with central, centrifugal, cicatricial alopecia (CCCA) in an attempt to define BZ integrity.

Improvement of tuberous sclerosis complex (TSC) skin tumors during long-term treatment with oral sirolimus

BACKGROUNDnOral mechanistic target of rapamycin inhibitors have been shown to reduce visceral tumor volume in patients with tuberous sclerosis complex (TSC).nnnOBJECTIVEnWe sought to evaluate the cutaneous response to oral sirolimus in patients with TSC and an indication for systemic treatment, including long-term effects.nnnMETHODSnA retrospective analysis of 14 adult patients with TSC prescribed sirolimus to treat lymphangioleiomyomatosis was performed. Serial photographs of angiofibromas, shagreen patches, and ungual fibromas taken before, during, and after the treatment period were blinded, then assessed using the Physician Global Assessment of Clinical Condition (PGA). Microscopic and molecular studies were performed on skin tumors harvested before and during treatment.nnnRESULTSnSirolimus significantly improved angiofibromas (median treatment duration 12 months; median PGA score 4.5 [range 1.5-5]; Wilcoxon signed rank test, P = .018) and shagreen patches (median treatment duration 10 months; median PGA score 4.5 [range 3.5-5]; Wilcoxon signed rank test, P = .039), whereas ungual fibromas improved in some patients (median treatment duration 6.5 months; median PGA score 4.66 [range 2.75-5]; Wilcoxon signed rank test, P = .109). Clinical, immunohistochemical, or molecular evidence of resistance was not observed (range 5-64 months of treatment).nnnLIMITATIONSnThis was a retrospective analysis limited to adult women with lymphangioleiomyomatosis.nnnCONCLUSIONnOral sirolimus is an effective long-term therapy for TSC skin tumors, particularly angiofibromas, in patients for whom systemic treatment is indicated.

Erythropoietin-driven proliferation of cells with mutations in the tumor suppressor gene TSC2

Lymphangioleiomyomatosis (LAM) is characterized by cystic lung destruction, resulting from proliferation of smooth-muscle-like cells, which have mutations in the tumor suppressor genes TSC1 or TSC2. Among 277 LAM patients, severe disease was associated with hypoxia and elevated red blood cell indexes that accompanied reduced pulmonary function. Because high red cell indexes could result from hypoxemia-induced erythropoietin (EPO) production, and EPO is a smooth muscle cell mitogen, we investigated effects of EPO in human cells with genetic loss of tuberin function, and we found that EPO increased proliferation of human TSC2-/-, but not of TSC2+/-, cells. A discrete population of cells grown from explanted lungs was characterized by the presence of EPO receptor and loss of heterozygosity for TSC2, consistent with EPO involvement. In LAM cells from lung nodules, EPO was localized to the extracellular matrix, supporting evidence for activation of an EPO-driven signaling pathway. Although the high red cell mass of LAM patients could be related to advanced disease, we propose that EPO, synthesized in response to episodic hypoxia, may increase disease progression by enhancing the proliferation of LAM cells.

A model system to analyse the ability of human keratinocytes to form hair follicles.

Earlier studies showed that dermal cells lose trichogenic capacity with passage, but studies on the effect of keratinocyte passage on human hair follicle neogenesis and graft quality have been hampered by the lack of a suitable model system. We recently documented human hair follicle neogenesis in grafted dermal‐epidermal composites, and in the present study, we determined the effects of keratinocyte passage on hair follicle neogenesis. Dermal equivalents were made with cultured human dermal papilla cells and were overlaid with either primary or passaged human keratinocytes to form dermal‐epidermal composites; these were then grafted onto immunodeficient mice. Superior hair follicle neogenesis was observed using early keratinocyte cultures. Characteristics such as formation of hair shafts and sebaceous glands, presence of hair follicles with features of anagen or telogen follicles, and reproducible hair and skin function parameters make this model a tool to study human hair follicle neogenesis and development.