Mesenchymal–epithelial interactions involving epiregulin in tuberous sclerosis complex hamartomas

Shaowei Li*, Fumiko Takeuchi*, Ji-an Wang*, Qingyuan Fan*, Toshi Komurasaki†, Eric M. Billings‡, Gustavo Pacheco-Rodriguez‡, Joel Moss‡, and Thomas N. Darling*§

*Department of Dermatology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4712; †Molecular Biology Laboratory, Molecular and Pharmacology Laboratories, Taisho Pharmaceutical Co., Ltd., 430-1 Yoshino-cho, Saitma-shi, Saitama 331-9530, Japan; and ‡Translational Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 10, Room 6D05, MSC 1590, Bethesda, MD 20892-1590

Communicated by Martha Vaughan, National Institutes of Health, Bethesda, MD, December 31, 2007 (received for review November 30, 2007) Patients with tuberous sclerosis complex (TSC) develop hamarto- Like other hamartomas, those in TSC skin contain abnormal mas containing biallelic inactivating mutations in either TSC1 or numbers of several types of cells. In the dermis, there are TSC2, resulting in mammalian target of rapamycin (mTOR) activa- increased numbers of large stellate fibroblasts, capillaries, and tion. Hamartomas overgrow epithelial and mesenchymal cells in dermal dendritic cells (6–9). The epidermis is acanthotic (i.e., TSC skin. The pathogenetic mechanisms for these changes had not thickened from increased numbers of keratinocytes in the spi- been investigated, and the existence or location of cells with nous layer). Acanthosis is pronounced in PFs and variable in AFs biallelic mutations (‘‘two-hit’’ cells) was unclear. We compared TSC (7, 8). The epidermis of treated AFs, several months after argon skin hamartomas (angiofibromas and periungual fibromas) with or CO2 laser surgery, no longer appears acanthotic (10, 11). normal-appearing skin of the same patient, and we observed more We sought to determine whether the overgrowth of epidermal proliferation and mTOR activation in hamartoma epidermis. Two- and dermal cells in TSC skin hamartomas was caused by hit cells were not detected in the epidermis. Fibroblast-like cells in second-hit mutations in the epidermis and dermis or by a the dermis, however, exhibited allelic deletion of TSC2, in both ‘‘two-hit’’ cell population that induced proliferation of neigh- touch preparations of fresh tumor samples and cells grown from boring cells. Second-hit mutations in more than one cell popu- TSC skin tumors, suggesting that increased epidermal proliferation lation have been observed in TSC renal angiomyolipomas. These MEDICAL SCIENCES and mTOR activation were not caused by second-hit mutations in tumors contain blood vessels, smooth muscle cells, and fat cells; the keratinocytes but by mesenchymal–epithelial interactions. loss of heterozygosity (LOH) at the TSC1 or TSC2 locus in all expression arrays, used to identify potential paracrine factors of these cell lineages has been reported (12, 13). However, it released by mesenchymal cells, revealed more epiregulin mRNA in plausible that one cell population could influence neighboring fibroblast-like angiofibroma and periungual fibroma cells than in cells, especially in light of the many important mesenchymal– fibroblasts from normal-appearing skin of the same patient. Ele- epithelial interactions involved in skin development (14), wound vation of epiregulin mRNA was confirmed with real-time PCR, and healing (15), and skin tumorigenesis (16). Disruptions in these increased amounts of epiregulin were demonstrated with interactions could contribute to the formation of hamartomas in immunoprecipitation. Epiregulin stimulated keratinocyte prolifer- TSC skin and other organs. ation and phosphorylation of ribosomal protein S6 in vitro. These Here, we report that fibroblast-like cells in TSC skin tumors, results suggest that hamartomatous TSC skin tumors are induced but not epidermal cells, showed allelic deletion of TSC2. Epi- by paracrine factors released by two-hit cells in the dermis and that regulin, an epidermal (EGF)-related growth fac- proliferation with mTOR activation of the overlying epidermis is an tor, is overexpressed in fibroblast-like cells grown from TSC skin effect of epiregulin. tumors and may induce epidermal changes. Results angiofibromas ͉ paracrine ͉ periungual fibromas ͉ TSC1 ͉ TSC2 Keratinocyte Proliferation and mTOR Activation in TSC Skin Tumors. Tissue sections were stained for nuclear antigen Ki-67 as a amartomas are benign growths composed of cells normally marker of cell proliferation and phospho-S6 as a marker of Hfound in an organ or tissue but in abnormal mixture or mTOR activation. The number of Ki-67-positive cells in the organization. Clues to the pathogenesis of hamartomas have epidermis of TSC AFs and PFs was clearly larger than that in the come from studies of inherited syndromes in which these lesions same patient’s normal-appearing skin (Fig. 1). Cells that were are characteristic (1). Patients with tuberous sclerosis complex positive for Ki-67 were seen mainly in the basal layer of the (TSC), an autosomal dominant tumor syndrome affecting Ϸ1in epidermis, with few apparent in the dermis. 10,000 individuals, develop hamartomas in multiple organs Few cells in the patient’s normal-appearing skin were positive because of a mutation that inactivates one allele of a tumor for phospho-S6. These cells were restricted almost entirely to a suppressor gene, either TSC1 or TSC2 (2). The protein products thin layer of cells comprising the granular layer of the epidermis; of these , hamartin and tuberin, respectively, form a complex that inhibits the mammalian target of rapamycin (mTOR) pathway, which regulates cell growth (3). TSC hamar- Author contributions: S.L. and T.N.D. designed research; S.L., F.T., J.-a.W., Q.F., G.P.-R., and tomas typically show a ‘‘second-hit’’ somatic mutation that T.N.D. performed research; T.K. contributed new reagents/analytic tools; S.L., F.T., J.-a.W., Q.F., E.M.B., G.P.-R., J.M., and T.N.D. analyzed data; and S.L., J.M., and T.N.D. wrote the inactivates the wild-type allele. Loss of functional hamartin/ paper. tuberin in TSC tumors leads to hyperactivation of mTOR The authors declare no conflict of interest. signaling and increased cell number and cell size. Data deposition: The data reported in this paper have been deposited in the Gene Approximately 90% of TSC patients develop skin hamartomas Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE9715). such as facial angiofibromas (AFs) and periungual fibromas §To whom correspondence should be addressed. E-mail: [email protected]. (PFs) (4). These tumors are benign but can be disfiguring or This article contains supporting information online at www.pnas.org/cgi/content/full/ cause bleeding and pain. Current treatments are surgical and 0712397105/DC1. have the potential to leave scarring (5). © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0712397105 PNAS ͉ March 4, 2008 ͉ vol. 105 ͉ no. 9 ͉ 3539–3544 Downloaded by guest on October 2, 2021 Fig. 1. The epidermis of TSC skin tumors shows increased staining for Ki-67 compared with normal-appearing skin. Sections of patient’s normal skin (A and D), angiofibromas (B and C), and periungual fibromas (E and F) from two TSC patients were stained with anti-Ki-67 antibody, with normal mouse IgG1 (C and F) as negative controls for A and B, and D and E, respectively. (Scale bars, 35 ␮m.) Results were similar with three angiofibromas and five periungual fibromas from five patients.

almost no cells in the dermis were positive for phospho-S6 (Fig. Two-Hit Cells Are Present in the Dermis, but Not the Epidermis, of TSC 2A). In contrast, the acanthotic epidermis of TSC skin tumors Skin Tumors. The pattern of staining for phospho-S6 in TSC skin was strongly positive for phospho-S6 (Fig. 2C). The dermis also tumors appeared consistent with the possibility that keratino- contained spindle- and stellate-shaped cells positive for cytes and/or scattered fibroblast-like cells in the dermis repre- phospho-S6 scattered between collagen bundles, with greater sented two-hit neoplastic cells. To identify these two-hit cells, numbers located adjacent to the epidermis. Acanthosis in TSC samples were analyzed for LOH at the TSC1 and TSC2 loci. LOH skin tumors was accompanied by both increased keratinocyte was not detected in DNA extracted from microdissected epi- proliferation and mTOR activation. dermis or dermis of three different AFs (data not shown). Because LOH in two-hit cells could be masked by cellular heterogeneity, we turned to interphase fluorescence in situ hybridization (I-FISH), which allows individual nuclei to be scored for allelic deletion of TSC1 or TSC2, based on the presence of only one signal per nucleus rather than the expected two signals per nucleus for each gene. Keratinocytes derived from three AFs did not show allelic deletion of TSC1 or TSC2 (Table 1); almost all nuclei showed two signals each for TSC1 and TSC2, with 0–4% showing only one signal. (In normal tissues, one signal is often observed in a small percentage of cells because of insufficient hybridization or overlapping signals.) Touch preparations of the dermal side of normal-appearing skin and cultured fibroblasts derived from normal-appearing skin from TSC patients showed similar patterns, with 0–8% showing one signal for TSC1 and 2–12% showing a single signal for TSC2. Among touch preparations or cells cultured from AFs and PFs only 0–8% showed one signal for TSC1, but 11–51% of nuclei had one signal for TSC2. These results are consistent with allelic deletion of TSC2 in a fraction of cells located in the dermis of AFs and PFs, but not in the keratinocytes associated with these tumors, which suggested that the epidermal changes in the tumor originated from effects of tumor mesenchymal cells.

Epiregulin mRNA Content of Fibroblast-Like Cells Derived from TSC Skin Tumors Is Greater Than That of the Patient’s Fibroblasts from Normal-Appearing Skin. RNA from fibroblast-like cells grown from four AFs, three PFs, and normal fibroblasts from four patients were analyzed by using Affymetrix GeneChips. In total, amounts of 22 probe sets representing 20 genes were increased, and 33 probe sets representing 28 genes were decreased by 3-fold or more (tumor vs. normal) in both AFs and PFs [supporting Fig. 2. Immunoreactivity for phosphorylated ribosomal protein S6 in the information (SI) Table 3]. The mRNA with the greatest mean epidermis and dermis of TSC skin tumors but not normal-appearing skin. Sections of normal-appearing skin (A and B) or periungual fibromas (C and D) elevation in AFs and PFs was epiregulin, which was 11.8-fold that were incubated with rabbit anti-phospho-S6 (1:200) (A and C) or control rabbit of the patient’s normal fibroblasts in AFs and 22.5-fold in PFs. IgG) (B and D) and stained with Vectastain ABC-alkaline phosphatase with Another mRNA that was highly overexpressed was MCP-1, Vector red substrate). Patterns of staining were similar in three periungual which was 9.5-fold the control level in AFs and 3.9-fold in PFs, fibromas and four angiofibromas from four patients. consistent with our earlier experiments using filter-based arrays

3540 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0712397105 Li et al. Downloaded by guest on October 2, 2021 Table 1. I-FISH analysis of TSC1 and TSC2 genes in TSC tumors Table 2. Gene expression of epiregulin in TSC skin tumor cells compared with normal skin fibroblasts from the same patient Percentage of single FISH signal Epiregulin, pg/18S rRNA, ng Tumor/normal Touch preparation Patient AF PF NL AF PF cells Cultured cells 2 1.92 44.0 0.30 6.4 147 Patient Tissue TSC1 TSC2 TSC1 TSC2 3 27.0 8.19 0.079 342 104 4 48.0 94.0 2.13 23 44 Keratinocytes 10 17.9 510 0.090 198 5,660 1NLNDND22 11 158 0.229 690 AF1 ND ND 2 4 12 68.9 0.120 574 AF2 ND ND 4 6 13 0.70 0.19 3.7 AF3 ND ND 0 2 14 2.25 0.023 98 2NLNDND06 15 74.2 0.49 151 AF ND ND 6 6 16 79.4 17.7 4.5 3NLNDND44 17 217 0.050 4,360 AF ND ND 2 4 18 16.9 36.3 0.032 522 1,120 PF ND ND 0 0 19 125 0.262 479 Fibroblasts 20 8.39 0.073 114 2 NL*† ND ND 4 8 21 16.5 0.215 77 AF*† ND ND 0 20 22 198 9.25 21 PF*† ND ND 0 18 23 8.27 0.080 103 3NL† ND ND 2 8 AF† 451226Epiregulin mRNA levels in normal or tumor cells from indicated patients 4 NL*† ND ND 0 10 were quantified by real-time PCR in duplicate. Results were normalized to 18S AF*† ND ND 6 20 rRNA, and the ratios of amount in tumor cells to that in normal fibroblasts PF1 2 19 2 28 from the same patient were calculated. PF2*† 422218 5 NL640 8 MEDICAL SCIENCES noprecipitation with antibodies against epiregulin, which yielded PF 2 19 0 24 immunoreactive of Ϸ5 kDa by using medium from 6 NL26012 fibroblast-like cells of TSC skin tumors but not from patients’ PF 2 11 0 12 normal-appearing skin (Fig. 3 and SI Fig. 6). The addition of 7NLNDND06 unlabeled recombinant epiregulin protein before immunopre- AF1 2 23 0 28 cipitation eliminated this band but not other, apparently non- AF2 2 32 2 17 specific bands. Incubation of cells with EGF did not measurably AF3 4 18 0 17 alter epiregulin release by TSC tumor cells or normal skin 8 NL042 2 fibroblasts (Fig. 3). Searchlight Protein Array analysis showed AF 2 20 ND ND that the levels of epiregulin in conditioned medium from PF cells 9NL06NDND of two patients were 12.8 and 43.6 pg/ml, respectively, whereas PF 2 30 0 35 the protein in conditioned medium from their normal TSC 10 NL*† 84012 fibroblasts was undetectable. Changes in levels of other EGF AF*† 818028 family members (EGF, TGF-␣, heparin-binding EGF-like PF1*† 232026 growth factor, and ) and keratinocyte growth fac- PF2 0 26 0 14 tor, if seen, were inconsistent (data not shown). 11 NL† 61008 AF1† 022014 AF2 2 20 2 18 12 NL*† ND ND 0 4 AF*† ND ND 0 18

NL, normal-appearing skin; AF, angiofibroma; PF, periungual fibroma, ND, not done. *Gene expression by Affymetrix array for cells in culture. †Epiregulin gene expression by real-time PCR for cells in culture.

(17). Epiregulin, an EGF family member, was investigated further because it appeared relevant to the observed epidermal changes. Amounts of epiregulin mRNA measured in samples from 17 patients using real-time PCR were 3.7- to 690-fold the paired controls in AF cells (n ϭ 13, P ϭ 0.001) and 4.5- to 5,660-fold in PF cells (n ϭ 9, P ϭ 0.008; Table 2). The addition Fig. 3. Immunoprecipitation of epiregulin from medium of TSC skin tumor of 100 nM rapamycin for 24 h did not significantly change cells. PF cells or fibroblasts from normal-appearing skin (NL) from the same TSC epiregulin mRNA levels (P ϭ 0.84; SI Table 4). patient were grown in 10% FBS/DMEM without or with 100 ng/ml EGF and labeled with [35S]methionine for 24 h. Proteins precipitated from conditioned medium with anti-epiregulin antibody or normal rabbit IgG were separated Release of Epiregulin Protein by Fibroblast-Like Cells from TSC Skin by SDS/PAGE to identify a band corresponding to epiregulin secreted by TSC 35 Tumors. After incubation of cells in medium containing [ S]me- skin tumor cells. Added recombinant epiregulin competed with radiolabeled thionine for 24 h, samples of medium were subjected to immu- endogenous epiregulin; absence of the band indicates antibody specificity.

Li et al. PNAS ͉ March 4, 2008 ͉ vol. 105 ͉ no. 9 ͉ 3541 Downloaded by guest on October 2, 2021 of hamartin or tuberin expression in fibroblastic interstitial but not epidermal cells (22, 23). Therefore, changes in epidermal cells overlying the tumor appear to the result of the influence of associated mesenchymal cells. Consistent with this view, we found that the release of epiregulin by fibroblast-like cells from TSC skin tumors was significantly greater than that by fibroblasts grown from normal skin of the same patients. Epiregulin is a member of the EGF family that includes EGF, TGF-␣, heparin-binding EGF-like growth factor, amphiregulin, and . Epiregulin was first purified from conditioned medium of the NIH 3T3/T7 mouse fibroblast-derived cell line Fig. 4. Effect of epiregulin on proliferation of human epidermal keratino- (24). Epiregulin is not produced by normal human fibroblasts, cytes. Cells were grown and treated with human recombinant epiregulin as but it is expressed by human fibroblasts immortalized by telom- described in Materials and Methods. Cell proliferation was quantified by the erase reverse transcriptase (25), fibroblastic cells in malignant amount of BrdU incorporation using chemiluminescence ELISA. Data are fibrous histiocytomas (26), and, as we show here, fibroblast-like means Ϯ SD of triplicate experiments. *, P Ͻ 0.01 versus control. cells from TSC skin tumors. Epiregulin is an autocrine or paracrine factor that is produced by many human cancers (27, 28). We propose that epiregulin released by fibroblast-like cells in Keratinocyte Proliferation and Phosphorylation of Ribosomal Protein TSC skin tumors stimulates epidermal cell proliferation and S6 Enhanced by Epiregulin. Recombinant epiregulin added in vitro acanthosis. This hypothesis is supported by in vitro studies to normal human keratinocytes stimulated cell proliferation in a presented here and others showing that epiregulin is mitogenic concentration-dependent manner (Fig. 4), with a maximum for keratinocytes (29–31) and stimulates phosphorylation of 1.9-fold increase in the rate of BrdU incorporation with 20 ng/ml. EGF receptor in keratinocytes (30) and by the in vivo demon- Treatment of keratinocytes with epiregulin increased phosphor- stration that application of recombinant epiregulin to murine ylation of ribosomal protein S6 within 10 min, which persisted at excisional wounds increased epidermal proliferation and thick- 1 h and decreased to basal levels after 24 h (Fig. 5 A and C). The ness (32). Epiregulin has also been implicated as a contributing epiregulin effect was concentration-dependent and maximal at factor to the epidermal changes in psoriasis because it is over- 100 ng/ml (Fig. 5 B and D). Epiregulin increased phosphoryla- expressed by acanthotic psoriatic epidermis (33). Other para- tion of p70S6K similar to ribosomal protein S6 (data not shown). crine factors may also participate in epidermal acanthosis in TSC The data indicate that epiregulin enhanced keratinocyte prolif- skin tumors, but we found that other EGF family members and eration and mTOR activation. keratinocyte growth factor were inconsistently, if at all, elevated in tumor-derived cells. Discussion The cause of increased epiregulin expression by fibroblast-like Increased epidermal thickness of AFs and PFs was associated cells derived from TSC skin tumors is unclear. It may not be with increased proliferation of keratinocytes and mTOR acti- related to mTOR activation resulting from TSC2 loss because vation. Although mTOR activation is characteristic of two-hit rapamycin treatment did not decrease epiregulin mRNA levels. populations in other TSC-related tumors (18–21), we found no Furthermore, treatment of normal fibroblasts with TSC2 siRNA evidence of second-hit mutations in the epidermal keratinocytes. dramatically decreased tuberin levels, but epiregulin mRNA Instead, allelic deletion of TSC2 was observed in fibroblast-like levels were only 1.5-fold that in cells treated with control cells from the AFs and PFs. These results are consistent with siRNA (S.L., Q.F., and T.N.D., unpublished observations). Recombinant EGF (100 ng/ml) increased the levels of epiregulin immunohistochemical studies of AFs and PFs that showed loss mRNA Ϸ50% in TSC skin tumor cells and normal fibroblasts (S.L. and T.N.D., unpublished observations), consistent with the work of others showing stimulation of epiregulin production through the EGFR pathway (30, 34, 35). This finding appears not to account for elevated levels of basal expression in tumors, however, because treatment of TSC skin tumor cells with AG1478, an EGFR inhibitor, or U0126, a MEK inhibitor, did not change epiregulin mRNA levels (S.L., J.-a.W., and T.N.D., unpublished observations). We note that epiregulin expression is directly regulated by the aryl-hydrocarbon receptor (36), one of the genes identified by using the arrays as overexpressed 2.1-fold in TSC tumor cells. The disorganized overgrowth of hamartomas is suggestive of abnormal continued expression of factors that stimulate growth early in development. Epiregulin is a plausible candidate for a ‘‘hamartic’’ factor in TSC because it is normally expressed early in development and exists in only a few adult cell lineages (24, Fig. 5. Effect of epiregulin on phosphorylation of S6 ribosomal protein in 37). Persistent expression of epiregulin by fibroblast-like cells in keratinocytes. After incubation for 48 h in keratinocyte-SFM (without added TSC skin tumors would be consistent with a less than normally EGF and bovine pituitary extract), cells were incubated without or with 100 differentiated fibroblast phenotype. In this regard, it is of interest ng/ml epiregulin for the indicated times (A and C) or without or with epiregu- that expression of epiregulin in the adult mouse cornea is lin at the indicated concentrations for1h(B and D) before separation of cell restricted to a stem cell population in the limbal epithelium (38). proteins by SDS/PAGE and reaction of Western blots with antibodies against S6 and phosphorylated S6. Shown are representative blots (A and B) and Hamartic factors may be expected to play other roles in tissue corresponding graphs from triplicate experiments (C and D), in which values morphogenesis. Investigation of the functions of epiregulin in are expressed as the means Ϯ SD of the ratios of phospho- to total S6 for organ formation is at an early stage, but it has been reported to treated (at each time or concentration) relative to that of control (time ϭ 0or be important in nephron morphogenesis (39). Epiregulin and its concentration ϭ 0). *, P Ͻ 0.05; **, P Ͻ 0.01 vs. control. receptors or downstream effectors, which appear to be involved

3542 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0712397105 Li et al. Downloaded by guest on October 2, 2021 in the formation of hamartomas in skin and possibly other organs (Applied Biosystems, Hs 00154995࿝AI), and values were expressed relative to affected by TSC, could be a therapeutic target for treatment of levels in the same cells of 18S rRNA by using assay-on-demand primers Hs this disease. 99999901࿝S1. Real-time PCR was performed by using the GeneAmp 5700 sequence detection system (Applied Biosystems). Materials and Methods Immunoprecipitation and Multiplex Assay. Epiregulin protein in conditioned Tumor Sample and Cell Culture. Patients meeting clinical criteria for diagnosis medium of TSC skin tumor cells was detected by using metabolic labeling and of TSC (40) were enrolled in protocol 00-H-0051 approved by the National immunoprecipitation as reported in ref. 30. Briefly, patient normal fibroblasts Heart, Lung, and Blood Institute/National Institutes of Health (NIH) Institu- or skin tumor cells (1 ϫ 106 cells per 10-cm dish) were labeled in 5 ml of tional Review Board. Samples of AFs, PFs, and normal-appearing skin were methionine-free DMEM containing 10% dialyzed FBS (Invitrogen) and 1 mCi obtained and bisected, with one portion used for routine pathology and the of [35S]methionine (Amersham Pharmacia Biotech). To some, 100 ng/ml EGF other used for frozen sections or cell culture. Fibroblasts from normal- was added at the beginning of the labeling. After 24 h, conditioned media appearing skin or fibroblast-like cells from AFs and PFs were isolated from were collected, and an aliquot (1.25 ml) of each medium was used for explants as described in ref. 41 and then cultured in DMEM supplemented with immunoprecipitation with rabbit anti-epiregulin IgG. The immunoprecipi- 10% FBS, 100 units/ml penicillin, and 100 ␮g/ml streptomycin. Keratinocytes tants were subjected to electrophoresis in 18% Tris-glycine gels (Invitrogen) from TSC skin samples were obtained by using standard methods (42). Kera- and analyzed by using the FLA-5100 Bio-Imaging System (Fujifilm Medical tinocytes were cultured in serum-free medium (keratinocyte-SFM; Invitro- System, Inc.). Epiregulin protein released by TSC skin tumor cells was quanti- gen), supplemented with EGF and bovine pituitary extract. For studies of the fied by Thermo Scientific SearchLight multiplex assay (Pierce) according to the effects of epiregulin on proliferation and cell signaling, keratinocytes from manufacturer’s procedures. foreskins of unidentified normal neonates were used (provided by Jonathan Vogel, National Cancer Institute, NIH). Cell Proliferation. Keratinocytes from neonatal human foreskins were added to 96-well Costar Black plates with clear bottoms (1 ϫ 104 cells per well). The Immunohistochemical Staining. Sections were incubated with mouse anti- next day, wells were washed with keratinocyte-SFM without bovine pituitary human Ki-67 monoclonal antibody (1 ␮g/ml, clone MIB-1; DakoCytomation), extract and human recombinant EGF, and cells were incubated for 24 h rabbit anti-phospho-S6 (1:200; Cell Signaling Technology), or source- and subtype-matched control IgG (DakoCytomation). Sections were stained with followed by incubation for 24 h more in the same medium containing the a Vectastain Elite ABC and 3,3Ј-diaminobenzidine peroxidase substrate indicated concentrations of recombinant epiregulin. Cells were labeled with ␮ kit or Vectastain ABC-alkaline phosphatase with Vector red substrate 10 M BrdU for 3 h, and incorporated BrdU was quantified by using a (Vector Laboratories) and counterstained with Mayer’s hematoxylin solution chemiluminescent ELISA kit (Roche), with chemiluminescence measured with (Sigma–Aldrich). a Fluoroskan Ascent FL (Thermo Labsystems).

Western Blot Analysis. Neonatal human foreskin keratinocytes were plated on I-FISH. Cells obtained from touch preparations or culture of TSC normal- MEDICAL SCIENCES ϫ 5 appearing skin and skin tumors were analyzed for allelic deletion of TSC1 and 60-mm dishes at 6 10 cells in keratinocyte-SFM. The next day, medium was TSC2 by using I-FISH as was described in ref. 43. At least 50 nuclei were scored changed to keratinocyte-SFM without EGF and bovine pituitary extract and for each sample. incubated for 24 h. Cells were treated with epiregulin as indicated and lysed in protein extraction buffer [20 mM Tris (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 20 mM NaF, 2.5 mM Na P O ,1mM␤-glycerophosphate, 1 mM benza- Gene Expression Array. Early passage cells (passages 3–5) were grown until 2 4 7 midine, 10 mM p-nitrophenyl phosphate, 1 mM phenylmethylsulfonyl fluo- 70–80% confluent. Total RNA was extracted by using an RNeasy minicolumn ride]. Equivalent samples of total proteins were separated in 10% (wt/vol) (Qiagen), and gene expression analysis was performed as described in the polyacrylamide gels and transferred to 0.45-␮m Invitrolon PVDF membranes Affymetrix expression analysis technical manual by using 10 ␮g of total RNA. (Invitrogen) before immunoblotting with 5 ␮g of anti-phospho-S6 ribosomal Fragmented, biotinylated cRNA was hybridized to U133A protein (Ser-235/236) or anti-S6 ribosomal protein antibodies (Cell Signaling), GeneChips (Affymetrix). Fluorescent probes were added, and intensities were horseradish peroxidase-conjugated anti-rabbit antibodies (GE Healthcare), quantified with a GeneArray scanner. Arrays that passed initial quality control and a SuperSignal West Pico chemiluminescence detection kit (Pierce). Band requirements were analyzed with Affymetrix MAS 5.1. A total of 11 arrays intensity was measured by using a Kodak Capture DC 290 imaging system from four patients were analyzed. Each PF and AF were first compared with (Eastman Kodak). the respective patient’s control cells from behind the ear or the back. Analysis of the intensities showed that differences between the back and ear locations were small compared with differences between controls and tumor tissue. Statistical Analysis. Epiregulin mRNA data were evaluated by using Wilcoxon Also, a comparison of results using normalization methods based on individ- signed-rank test. Proliferation (chemiluminescence values) and S6 phosphor- ual controls vs. grouped controls showed little effect (with 994 of 1,180 ylation (ratio of phospho- to total S6 on a log scale) were assessed by using possible probe sets in common). Probe sets that were identified as ‘‘present or two-way ANOVA with the primary variable epiregulin or time adjusted for marginal’’ by MAS 5.1 were used for subsequent analysis. Lists of probe sets differences in baseline level among experiments, followed by Dunnett t test were constructed for evaluation including those with threefold change and/or to compare each treatment group with control. Significance was defined as Ͻ statistically significant with a false discovery rate Ͻ1% by using a Benjamini– P 0.05. Hochberg multiple testing correction. ACKNOWLEDGMENTS. We thank the Tuberous Sclerosis Alliance and The LAM Foundation for patient referral. We also thank Catherine Jozwik for the Real-Time PCR. Tumor cells or normal fibroblasts grown from AFs, PFs, or radiolabeling labeling of the cells. This work was supported by a Clinical normal skin were incubated in 1% FBS/DMEM with or without 100 nM Scientist Development Award from the Doris Duke Charitable Foundation and rapamycin for 24 h before isolation of total RNA from the cells by RNeasy mini National Institutes of Health (NIH) Grant 1 R01 CA100907 (to T.N.D.). G.P.-R. kit (Qiagen). Epiregulin mRNA levels were quantified by real-time PCR using and J.M. were supported by the Intramural Research Program, National Heart, the TaqMan universal master mix with assay-on-demand epiregulin primers Lung, and Blood Institute, NIH.

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