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Upregulation of the WNT pathway in tuberous sclerosis-associated subependymal giant cell astrocytomas
Brain & Development 29 (2007) 273–280 www.elsevier.com/locate/braindev
Original article
Upregulation of the WNT pathway in tuberous sclerosis-associated subependymal giant cell astrocytomas Jaroslaw Jozwiak a, Katarzyna Kotulska b, Wieslawa Grajkowska c, Sergiusz Jozwiak b, Wojciech Zalewski a, Monika Oldak a, Magdalena Lojek a, Kamila Rainko a, Radosław Maksym a, Maciej Lazarczyk a, Piotr Skopinski a, Pawel Wlodarski a,* a
Department of Histology and Embryology, Center for Biostructure Research, Medical University of Warsaw, Warsaw, Poland b Department of Neurology and Epileptology, The Children’s Memorial Health Institute, Warsaw, Poland c Department of Pathology, The Children’s Memorial Health Institute, Warsaw, Poland Received 11 July 2006; received in revised form 5 September 2006; accepted 11 September 2006
Abstract Tuberous sclerosis (TS), autosomal dominant disorder manifested by the formation of usually benign tumors in the brain, heart, kidneys and skin, results from an inactivating mutation in one of two tumor suppressor genes TSC1 or TSC2. Protein products of these genes, hamartin and tuberin, respectively, have been shown to participate in the mTOR pathway controlling translation of approx. 10–15% of all proteins. In the current paper, we aimed at verifying whether hamartin and tuberin may also be implicated in the control of gene transcription. Very recently it has been hypothesized that the pathway triggered by WNT, one of embryonic growth factors involved in cell differentiation and migration, could be disturbed in TS. In order to test this hypothesis we evaluated samples of four subependymal giant cell astrocytomas (SEGAs), brain tumors developing in the progress of TS. We found that b-catenin, transcription factor and mediator of WNT pathway activity is indeed present and active in SEGAs. mRNA transcripts for c-Myc and N-Myc, proteins whose transcription is regulated by b-catenin, were upregulated in two of four SEGAs, while cyclin D1 mRNA was significantly higher in three SEGAs. At the same time, c-Myc and N-Myc proteins were detected in the same two samples. Thus, we show for the first time that aberrant WNT signaling may contribute to the pathogenesis of TS-associated SEGAs. 2006 Elsevier B.V. All rights reserved. Keywords: Tuberous sclerosis; WNT pathway; b-Catenin; SEGA
1. Introduction Tuberous sclerosis (TS) is an autosomal dominant disease associated with mutation of one of two tumor suppressor genes, encoding hamartin (TSC1) or tuberin (TSC2). Both proteins are ubiquitous, form an intracellular complex (TSC1/TSC2 complex) and together exert a GTP-ase activating protein (GAP) activity towards Ras-homologue enriched in brain (Rheb) [1]. In consequence, Rheb is unable to activate mTOR (mammalian *
Corresponding author. E-mail address: [email protected] (P. Wlodarski).
0387-7604/$ - see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.braindev.2006.09.009
target of rapamycin) and promote protein translation. In disease states, when hamartin or tuberin are unfunctional, Rheb is released from TSC1/TSC2 complex inhibition and activates mTOR. Precise mechanism of mTOR activation by Rheb has been described very recently [2]. mTOR-dependent proteins are synthesized under strict control of translation. Two substrates of mTOR, p70 S6K1 and 4E-BP1, are phosphorylated and confer the translational signal leading to ribosome recruitment and translation initiation [3,4]. Thus, mTOR is often described as a central regulator of protein synthesis. Stimuli concerning internal cell status, i.e., low energy
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levels, hypoxia or nutrient availability, are transmitted through the mediation of specific proteins (Akt, Erk, REDD, AMPK) to mTOR. Interestingly, it has been elucidated recently, that all of those proteins do not act directly on mTOR, but modulate the activity of TSC1/TSC2 complex [5]. WNTs (Wingless) are a family of embryonic growth factors that coordinate gene expression and cell-to-cell interactions during embryogenesis [6]. It has been found that in adult tissues WNT signals lead to mitogenic stimulation and cell differentiation and thus, are often implicated in cancer development. Impairment of the WNT signaling appears to be associated with neurodegenerative disorders. In the so-called canonical pathway, kinases induced by WNT control gene transcription through b-catenin, protein participating in the formation of the cell-to-cell attachments and, more importantly, functioning as a transcription factor for several genes which regulate cell cycle, such as cyclin D1 or c-Myc. WNT molecules bind to a class of transmembrane receptors encoded by the Frizzled genes [7,8]. Frizzled molecules constitute a class of seven transmembrane proteins with a cysteine-rich domain (CRD) at the N-terminus. WNTs bind directly to CRD, however signal propagation requires participation of one more transmembrane molecule LRP (LDL receptor related protein). WNT, Fz and LRP form a trimeric membrane complex able to confer intercellular signal. Precise mechanism of signal propagation is unknown, however it is assumed that WNT receptor activation leads to phosphorylation of Dishevelled (Dvl) protein which, after associating with the scaffolding molecule of Axin, phosphorylates and inhibits GSK-3b (glycogen synthase kinase 3b)-mediated phosphorylation of b-catenin at S33 and S37. Phosphorylated b-catenin is ubiquitinated by SCFbTRCP, a complex of E3 ubiquitin ligase, and is destined for degradation by proteasomes. If, however, b-catenin remains unphosphorylated, it migrates to the nucleus and recruits transcription factors such as TCF (T-cell factor) and LEF (lymphoid enhancer factor) and activates transcription of its target genes [9,10]. Significance of the WNT pathway is postulated in colon cancer or melanoma, but also in neurological tumors like medulloblastoma, the most common primary central nervous system tumor in children [11]. As deregulation of the WNT pathway is often found in neuropathological conditions, it is postulated that the research on precise regulating mechanisms should contribute to establishing treatment modalities for illnesses like Alzheimer’s disease, as well as in neuropsychiatric diseases or retinal degenerations [12]. Both pathways, mTOR and WNT, have a common link: TSC1/TSC2 complex. Mak and colleagues [13] showed that this complex is involved in phosphorylation
and degradation of b-catenin. In the absence of wildtype TSC1 or TSC2 b-catenin accumulates in the cytoplasm and translocates to the nucleus, activating transcription of genes like c-Myc, N-Myc or cyclin D1. Indeed, cyclin D1, playing the key regulatory role during the G0/G1 transition of the cell cycle [14], was found elevated in tuberous sclerosis-associated renal tumors in the Eker rats [15]. This elevation was independent from mTOR activation, as shown by the treatment with mTOR inhibitor, rapamycin. In such a setting, cyclin D1 expression was not decreased, although cell proliferation was significantly inhibited. In the present paper, we evaluated for the first time status of the WNT pathway in subependymal giant cell astrocytomas (SEGAs), brain tumors of patients diagnosed with tuberous sclerosis. We tested b-catenin phosphorylation as well as the presence of gene transcripts controlled by b-catenin: c-Myc, N-Myc and cyclin D1. We found that c-Myc is upregulated strongly in two and moderately in the remaining two SEGAs. c-Mycabundant SEGAs showed also moderate presence of N-Myc, which resulted from increased transcription of c-Myc and N-myc genes, respectively, as shown by RT-PCR. Surprisingly, cyclin D1 protein was not found in any SEGA, although its mRNA was detected in all four cases.
2. Materials and methods 2.1. Tissue samples Samples of four SEGAs from four different patients as well as control brain tissue were excised during elective surgery and retrieved from the Department of Pathology, Children’s Memorial Hospital, Warsaw, Poland. Tuberous sclerosis patients were clinically diagnosed according to the criteria of Roach [16]. Control brain tissue consisted of periventricular regions of a patient operated on for epilepsy, with no symptoms of TS. As control for WNT stimulation we used hemimegalencephaly sample collected from a patient operated on because of intractable epilepsy, as well as resected medulloblastoma. The study has been approved by the Ethics Committee of the Children’s Memorial Health Institute, Warsaw, Poland. All patients and their parents gave their informed consent prior to their inclusion into the study. 2.2. Light microscopy Tissue specimens were fixed in 10% buffered formalin, at room temperature for 24 h, embedded in paraffin and processed according to routine technique of light microscopy. Sections 5–8 lm thick were stained with hematoxylin and eosin.
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2.3. Protein samples Tissues were homogenized in tissue grinder with RIPA lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA, 0.1% SDS) with 50 mM sodium fluoride and 1 mM sodium orthovanadate, supplemented with 1· Complete Protease Inhibitor (Roche, Indianapolis, IN, USA) and Phosphatase Inhibitor Cocktail I (Sigma–Aldrich, St. Louis, MO, USA). In order to minimize differences in sample preparation procedure, all the samples were processed simultaneously, in standardized conditions. Protein content was measured using Bradford protein assay kit (Bio-Rad, Hercules, CA, USA). Lysates were stored at 80C. 2.4. Western blot Twenty micrograms of protein extracted from tissues were resolved on 12% SDS–PAGE. Afterwards, the gels were transferred onto PVDF membranes. Quality of electrophoresis and transfer was evaluated with Ponceau S staining. Blots were blocked with 5% non-fat dry milk in TBST (Tris buffered saline, 0.05% Tween) and incubated with respective primary and secondary (HRP-conjugated) antibodies. Membranes were washed in TBST buffer and proteins were detected by West Pico chemiluminescence substrate (Pierce, Rocford, IL, USA). 2.5. Immunohistochemistry Immunohistochemical studies were performed on formalin-fixed, paraffin-embedded specimens. Sections of 10 lm were mounted on slides covered with NovoBond (Novocastra, United Kingdom), air-dried for 24 h and deparaffinized through xylene and graded alcohols to PBS (phosphate-buffered saline). To facilitate antibody bounding, the high-temperature antigen unmasking method using citrate buffer was applied. Unspecific bounding sites were blocked with 10% BSA (bovine serum albumin) and 10% goat serum (both from Sigma–Aldrich). Sections were then treated with primary antibodies. In the case of polyclonal antibodies, diluted non-immune serum was used instead of primary antibody, as negative control. For monoclonal antibodies, diluted IgG of relevant isotype was used as negative control. The specimens were kept in humid chamber at 37 C for 1 h. After triple rinsing in PBS, the mixture of secondary antibodies was applied. Staining was performed in moist chamber, at room temperature, for 2 h. Then, after extensive rinsing in PBS, the slides were covered with VectaShield (Vector, USA) and coverslipped. The specimens were examined under the confocal microscope Olympus Fluoview (Olympus, Japan). The pictures of both excitation wavelengths were merged for analysis and data presentation. The semiquantitative analysis
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was done by two independent observers, according to the following criteria: Intensity o staining No signal Weak signal Moderate signal Bright cells
Percent of positive cells 0
0–5
5–25
25–50
>50
+
+ ++
+ ++ +++
++ +++ +++
2.6. Antibodies Antibodies against: b-catenin, phospho-b-catenin (T 33), c-Myc, N-Myc, cyclin D1 and secondary antibodies (HRP-goat anti-rabbit or HRP-bovine anti-mouse) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). For immunohistochemistry, secondary antibodies conjugated with AlexaFluor 488 or AlexaFluor 568 were obtained from Molecular Probes Inc. (Eugene, OR, USA). 2.7. RT-PCR Total RNA was isolated using Trizol reagent (Gibco BRC, Carlsbad, CA, USA) according to manufacturer’s instructions. The RNA pellet was suspended in DEPC water and RNA concentration was assessed by optical density. Two micrograms of RNA was used in reverse transcriptase reaction using oligo(dT)15 primer and M-MLV from Invitrogen (San Diego, CA, USA) according to the protocol of the producer. Expression of GAPDH mRNA was determined by PCR in restricted number of cycles. Twenty cycles of 95 C-3000 , 57 C-3000 and 72 C-3000 with 1 U/25 ll Taq polymerase (Invitrogen), 75 lM of each dNTP and 0.5 lM of each primer raised specific product which was analyzed on 2% TAE agarose gel stained with ethidium bromide. Primers for GAPDH amplifications were: GAPDH forward: 5 0 -AGG TCG GAG TCA ACG GAT TT and GAPDH reverse: 5 0 -CAG CAG AGG GGG CAG AGA TG. After cDNA was normalized to GAPDH, the expression of c-Myc and N-Myc mRNA was evaluated in the similar PCRs. As the efficiency of these reactions was significantly lower, the products visualized in UV were obtained after 30 cycles of 95 C-3000 , 58 C-3000 and 72 C-3000 . Primers for c-Myc, N-Myc and cyclin D1 were as follows: c-Myc forward: 5 0 -ACT CGG TGC AGC CGT ATT TCT ACT, c-Myc reverse: 5 0 -TCT GAC ACT GTC CAA CTT GAC CCT; N-Myc forward: 5 0 -ACA GAC TGT AGC CAT CCG AGG ACA, N-Myc reverse: 5 0 -GAG GTC TGG GTT CTT GCA GAT CAT; cyclin D1 forward: 5 0 -CCC TCG GTG TCC TAC TTC AAA TGT, cyclin D1 reverse: 5’-TGA TCT GTT TGT TCT CCT CCG CCT.
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All primers were synthesized by the Institute of Biochemistry and Biophysics of Polish Academy of Sciences.
3. Results 3.1. SEGAs show typical cell composition with characteristic giant cells Light microscopy showed typical components: spindle-shaped fibrillated cells, polygonal cells with eosinophilic cytoplasm and characteristic giant cells with eccentric vesicular nuclei. Small areas of necrosis (like the one shown in Fig. 1) were seen occasionally.
3.2. b-Catenin levels are increased in SEGAs Western blot of protein extracts from SEGAs demonstrated elevated levels of b-catenin. In SEGA1 and SEGA4 b-catenin was significantly elevated compared to normal human brain, while SEGA2 and SEGA3 showed moderate amount of b-catenin. However, phosphorylation of b-catenin at Ser33, an inevitable step towards its ubiquitination and degradation, was not detected in any of the tested SEGA samples, while it was present in normal brain (Fig. 2). Extracts from human medulloblastoma and hemimegalencephaly, where activation of the WNT pathway is documented [17,18] served as positive controls. In our experiments the presence of b-catenin and no phosphorylation of this protein were detected in both positive control samples.
Fig. 1. Histopathological images of SEGAs. (A, SEGA1) and (B, SEGA2): numerous giant cells with eccentric nuclei; (C, SEGA3): prominent nuclear pleomorphism in a giant cell close to the center of the image; (D, SEGA4): focus of necrosis in the middle of the photo. Bars indicate 50 lm.
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staining in most cells composing the tumor, including typical giant cells. In the majority of cells staining distribution in cytoplasm and nucleus was homogenous (see example in Fig. 3). Control normal human brain samples did not show the presence of b-catenin in the nucleus. 3.4. Detection of protein products of c-Myc, N-Myc and cyclin D1
Fig. 2. Western blot showing reaction with antibodies against WNT pathway-associated proteins. The amount of proteins in lysates was normalized according to anti-actin antibody reaction (not shown). Hemimegalencephaly samples were used as positive control for the activation of the WNT pathway.
3.3. Nuclear translocation of b-catenin Hypophosphorylated b-catenin translocates to the nucleus and fulfils its role as a transcription factor [19]. Fluorescence microscopy demonstrated b-catenin
In order to confirm the activating function of b-catenin, we evaluated by Western blot and immunohistochemistry the expression of three proteins, whose transcription is known to be regulated by the WNT pathway: cyclin D1, c-Myc and N-Myc. Western blot showed strong upregulation of c-Myc in two of four SEGAs (SEGA2 and SEGA3) while in the remaining two cases c-Myc was present at the moderate level. NMyc protein levels corresponded with this result, although the expression of N-Myc was moderate in SEGA2 and SEGA3, and weak in SEGA1 and SEGA4. Surprisingly, cyclin D1 production was not upregulated in any of the SEGAs. The above results were confirmed by immunofluorescent staining (Table 1 and Fig. 3). Presented weakly stained giant cell was an exception in overall negative staining, as evaluated according to our criteria. c-Myc protein was detected at a similar, moderate level in SEGAs 2–4, while it was weak in SEGA1. Also N-Myc was detected, although in this case reaction was significantly weaker. Notably, all three antibodies, including anti-cyclin D1 antibody that showed very weak or no reaction, detected a subpopulation of giant cells with moderate
Fig. 3. Representative immunofluorescent staining of SEGA1 and SEGA2. Original magnification 400·. Arrows show giant cells stained with respective antibodies. Bars link photographs taken under the same visual field. Note homogeneous distribution of b-catenin seen in a giant cell of SEGA2. The same cell demonstrates cytoplasmic staining for N-Myc. Very weak cyclin D1 staining seen in an individual giant cell (see magnification), was classified as negative result, according to our criteria (see Section 2), whereas broader positive staining was detected in SEGA2. Control positive staining with all antibodies (including p-b-catenin and cyclin D1) has been performed with respective normal and tumor tissues, confirming correct detection of these antibodies (results not shown).
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Table 1 Semi-quantitative results of immunohistochemistry of SEGA tissue samples with use of respective antibodies; : no reaction; +: weak reaction; ++: moderate reaction; +++: strong reaction
b-Catenin p-b-catenin (Ser33) Cyclin D1 c-Myc N-Myc
SEGA1
SEGA2
SEGA3
SEGA4
++ +
+ + ++ +
+ + ++ +
+++ + ++ +
Positive control for p-b-catenin (not shown) demonstrated strong cytoplasmic staining.
to strong staining (Fig. 3). Thus, in comparison to normal brain, where cyclin D1 accumulation was not detected, in SEGAs cyclin D1 was only found in a limited number of giant cells. 3.5. Transcription of c-Myc, N-Myc and cyclin D1 Next, we made an attempt to determine whether higher levels of c-Myc protein were accompanied by upregulated transcription. For this purpose, reverse transcription-polymerase chain reaction (RT-PCR) was performed to determine the expression of mRNA for c-Myc, N-Myc and cyclin D1. The reaction showed increased transcription of c-Myc and N-Myc genes in SEGA2 and SEGA3, while the amount of transcripts in SEGA1 and SEGA4 remained on the level of healthy control (Fig. 4). To our surprise, in spite of lack of cyclin D1 protein product, we found its mRNA in all four SEGAs. RT-PCR on positive control (hemimegalencephaly) showed accumulation of mRNA for all three WNT-dependent genes.
Fig. 4. RT-PCR of SEGAs and control samples. Specific bands are shown. cDNAs used for the reaction were normalized according to GAPDH level.
4. Discussion TSC1/TSC2 complex is known to regulate protein translation through its participation in the mTOR pathway. As much as 15% of all proteins in a cell may be regulated by this mechanism [20]. There are, however, numerous age-related differences between different types of TS-associated tumors that cannot be explained by upregulation of mTOR-related translation alone. For example, cardiac rhabdomyomas are detected during embryogenesis and tend to regress after birth, while renal angiomyolipomas (AMLs) develop with age. Also, all three types of TS brain lesions (subependymal nodules, subependymal giant cell astrocytoma and cortical tubers) contain giant cells postulated to result from disturbed cell migration and differentiation. Giant cells have been tested for astrocytic and neuronal markers and found to express them at different levels (for a review see [21]). For these reasons we hypothesized that embryonic growth factors like WNT may contribute to the clinical picture of TS-associated tumors. Positive correlation between TSC2 mutation and amount of b-catenin transcription products has been postulated before. Cyclin D1 mRNA level was demonstrated to be upregulated in giant cells of cortical tubers microdissected from TS patients [22]. It has also been noted that antisense inhibition of tuberin in rat fibroblasts results in increased amount of cyclin D1 protein [23]. Very recently, Mak and collaborators [24] evaluated TS-related AMLs and lymphangioleiomyomatosis (LAM) from the point of view of WNT pathway activation and demonstrated that the tumors contain increased amount of b-catenin-dependent proteins: cyclin D1 and connexin 43. Our evaluation of the WNT pathway products in human brain tumors associated with tuberous sclerosis generally extends the research of Mak and colleagues [24] performed on human AML and LAM sections. There are however some differences in the amount of observed b-catenin-dependent proteins. According to our expectations, c-Myc and N-Myc mRNA was found in two of four SEGAs, and cyclin D1 mRNA was present at variable levels in all four samples. On the protein level however, c-Myc and N-Myc were prominent in the same SEGAs that revealed respective mRNA transcripts, while cyclin D1 did not appear in Western blot, and in situ immunostaining showed very scant reaction. It is worth stressing that all three antibodies against b-catenin-dependent proteins demonstrated positive staining in a population of giant cells, typical for the development of SEGA. Histological composition of both types of tumors, AML and SEGA, may contribute to our understanding of the differences in cyclin D1 presence and/or distribution. While AMLs are composed of varying amounts of thick-walled blood vessels, mature adipose tissue and smooth muscles [25], SEGAs
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contain three types of cells: astrocytes, dysmorphic neurons and characteristic giant cells [26]. Differential expression of cell cycle-related cyclin D1 in both types of lesions may also reflect the rate of their development: SEGAs are generally slowly growing neoplasms [27], while AMLs have a tendency to develop more rapidly [28]. One more explanation of the discrepancy in the amount of cyclin D1 mRNA and protein seems to be tempting. In 2004 Koziczak and Hynes [29] evaluated the cooperation of ErbB2 and fibroblast growth factor receptor-4 (FGFR-4) in the regulation of cyclin D1 production. The authors found that FGFR-4, triggering the Erk pathway, is responsible for the control over cyclin D1 transcription, while ErbB2, acting through Akt, is responsible for the increase in cyclin D1 translation. In our recent paper [30], performed on the same four SEGA samples, we have detected strong upregulation of the Erk pathway in all four SEGAs, and weak upregulation of Akt only in SEGAs 3 and 4. Thus, high levels of cyclin D1 mRNA in all tested SEGAs could result from high Erk activity, while low amount of the protein may be explained by weak Akt phosphorylation. Mak and colleagues [24] determined that the distribution of b-catenin staining within the tumors was variable and that b-catenin activation may be limited to the outer limits of the tumor. SEGA samples tested in this study were not collected from the outer part of tumors, which may additionally explain discrepancy with the results of Mak. It is noteworthy, that immunohistochemistry revealed nuclear staining of b-catenin in our samples. According to Eberhart and colleagues [19], active form of b-catenin implies activation of the WNT signaling pathway. The authors examined 48 glial and meningeal CNS tumors, all of which were negative for nuclear b-catenin, finding this type of reaction only in medulloblastoma samples, where participation of the WNT pathway is well-documented. Our results demonstrate that development of SEGA is accompanied by the activation of the WNT pathway, leading to transcription and overproduction of cell cycle-regulating proteins like c-Myc or N-Myc. However, precise mechanism and significance of this finding requires further research.
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Original article
Upregulation of the WNT pathway in tuberous sclerosis-associated subependymal giant cell astrocytomas Jaroslaw Jozwiak a, Katarzyna Kotulska b, Wieslawa Grajkowska c, Sergiusz Jozwiak b, Wojciech Zalewski a, Monika Oldak a, Magdalena Lojek a, Kamila Rainko a, Radosław Maksym a, Maciej Lazarczyk a, Piotr Skopinski a, Pawel Wlodarski a,* a
Department of Histology and Embryology, Center for Biostructure Research, Medical University of Warsaw, Warsaw, Poland b Department of Neurology and Epileptology, The Children’s Memorial Health Institute, Warsaw, Poland c Department of Pathology, The Children’s Memorial Health Institute, Warsaw, Poland Received 11 July 2006; received in revised form 5 September 2006; accepted 11 September 2006
Abstract Tuberous sclerosis (TS), autosomal dominant disorder manifested by the formation of usually benign tumors in the brain, heart, kidneys and skin, results from an inactivating mutation in one of two tumor suppressor genes TSC1 or TSC2. Protein products of these genes, hamartin and tuberin, respectively, have been shown to participate in the mTOR pathway controlling translation of approx. 10–15% of all proteins. In the current paper, we aimed at verifying whether hamartin and tuberin may also be implicated in the control of gene transcription. Very recently it has been hypothesized that the pathway triggered by WNT, one of embryonic growth factors involved in cell differentiation and migration, could be disturbed in TS. In order to test this hypothesis we evaluated samples of four subependymal giant cell astrocytomas (SEGAs), brain tumors developing in the progress of TS. We found that b-catenin, transcription factor and mediator of WNT pathway activity is indeed present and active in SEGAs. mRNA transcripts for c-Myc and N-Myc, proteins whose transcription is regulated by b-catenin, were upregulated in two of four SEGAs, while cyclin D1 mRNA was significantly higher in three SEGAs. At the same time, c-Myc and N-Myc proteins were detected in the same two samples. Thus, we show for the first time that aberrant WNT signaling may contribute to the pathogenesis of TS-associated SEGAs. 2006 Elsevier B.V. All rights reserved. Keywords: Tuberous sclerosis; WNT pathway; b-Catenin; SEGA
1. Introduction Tuberous sclerosis (TS) is an autosomal dominant disease associated with mutation of one of two tumor suppressor genes, encoding hamartin (TSC1) or tuberin (TSC2). Both proteins are ubiquitous, form an intracellular complex (TSC1/TSC2 complex) and together exert a GTP-ase activating protein (GAP) activity towards Ras-homologue enriched in brain (Rheb) [1]. In consequence, Rheb is unable to activate mTOR (mammalian *
Corresponding author. E-mail address: [email protected] (P. Wlodarski).
0387-7604/$ - see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.braindev.2006.09.009
target of rapamycin) and promote protein translation. In disease states, when hamartin or tuberin are unfunctional, Rheb is released from TSC1/TSC2 complex inhibition and activates mTOR. Precise mechanism of mTOR activation by Rheb has been described very recently [2]. mTOR-dependent proteins are synthesized under strict control of translation. Two substrates of mTOR, p70 S6K1 and 4E-BP1, are phosphorylated and confer the translational signal leading to ribosome recruitment and translation initiation [3,4]. Thus, mTOR is often described as a central regulator of protein synthesis. Stimuli concerning internal cell status, i.e., low energy
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levels, hypoxia or nutrient availability, are transmitted through the mediation of specific proteins (Akt, Erk, REDD, AMPK) to mTOR. Interestingly, it has been elucidated recently, that all of those proteins do not act directly on mTOR, but modulate the activity of TSC1/TSC2 complex [5]. WNTs (Wingless) are a family of embryonic growth factors that coordinate gene expression and cell-to-cell interactions during embryogenesis [6]. It has been found that in adult tissues WNT signals lead to mitogenic stimulation and cell differentiation and thus, are often implicated in cancer development. Impairment of the WNT signaling appears to be associated with neurodegenerative disorders. In the so-called canonical pathway, kinases induced by WNT control gene transcription through b-catenin, protein participating in the formation of the cell-to-cell attachments and, more importantly, functioning as a transcription factor for several genes which regulate cell cycle, such as cyclin D1 or c-Myc. WNT molecules bind to a class of transmembrane receptors encoded by the Frizzled genes [7,8]. Frizzled molecules constitute a class of seven transmembrane proteins with a cysteine-rich domain (CRD) at the N-terminus. WNTs bind directly to CRD, however signal propagation requires participation of one more transmembrane molecule LRP (LDL receptor related protein). WNT, Fz and LRP form a trimeric membrane complex able to confer intercellular signal. Precise mechanism of signal propagation is unknown, however it is assumed that WNT receptor activation leads to phosphorylation of Dishevelled (Dvl) protein which, after associating with the scaffolding molecule of Axin, phosphorylates and inhibits GSK-3b (glycogen synthase kinase 3b)-mediated phosphorylation of b-catenin at S33 and S37. Phosphorylated b-catenin is ubiquitinated by SCFbTRCP, a complex of E3 ubiquitin ligase, and is destined for degradation by proteasomes. If, however, b-catenin remains unphosphorylated, it migrates to the nucleus and recruits transcription factors such as TCF (T-cell factor) and LEF (lymphoid enhancer factor) and activates transcription of its target genes [9,10]. Significance of the WNT pathway is postulated in colon cancer or melanoma, but also in neurological tumors like medulloblastoma, the most common primary central nervous system tumor in children [11]. As deregulation of the WNT pathway is often found in neuropathological conditions, it is postulated that the research on precise regulating mechanisms should contribute to establishing treatment modalities for illnesses like Alzheimer’s disease, as well as in neuropsychiatric diseases or retinal degenerations [12]. Both pathways, mTOR and WNT, have a common link: TSC1/TSC2 complex. Mak and colleagues [13] showed that this complex is involved in phosphorylation
and degradation of b-catenin. In the absence of wildtype TSC1 or TSC2 b-catenin accumulates in the cytoplasm and translocates to the nucleus, activating transcription of genes like c-Myc, N-Myc or cyclin D1. Indeed, cyclin D1, playing the key regulatory role during the G0/G1 transition of the cell cycle [14], was found elevated in tuberous sclerosis-associated renal tumors in the Eker rats [15]. This elevation was independent from mTOR activation, as shown by the treatment with mTOR inhibitor, rapamycin. In such a setting, cyclin D1 expression was not decreased, although cell proliferation was significantly inhibited. In the present paper, we evaluated for the first time status of the WNT pathway in subependymal giant cell astrocytomas (SEGAs), brain tumors of patients diagnosed with tuberous sclerosis. We tested b-catenin phosphorylation as well as the presence of gene transcripts controlled by b-catenin: c-Myc, N-Myc and cyclin D1. We found that c-Myc is upregulated strongly in two and moderately in the remaining two SEGAs. c-Mycabundant SEGAs showed also moderate presence of N-Myc, which resulted from increased transcription of c-Myc and N-myc genes, respectively, as shown by RT-PCR. Surprisingly, cyclin D1 protein was not found in any SEGA, although its mRNA was detected in all four cases.
2. Materials and methods 2.1. Tissue samples Samples of four SEGAs from four different patients as well as control brain tissue were excised during elective surgery and retrieved from the Department of Pathology, Children’s Memorial Hospital, Warsaw, Poland. Tuberous sclerosis patients were clinically diagnosed according to the criteria of Roach [16]. Control brain tissue consisted of periventricular regions of a patient operated on for epilepsy, with no symptoms of TS. As control for WNT stimulation we used hemimegalencephaly sample collected from a patient operated on because of intractable epilepsy, as well as resected medulloblastoma. The study has been approved by the Ethics Committee of the Children’s Memorial Health Institute, Warsaw, Poland. All patients and their parents gave their informed consent prior to their inclusion into the study. 2.2. Light microscopy Tissue specimens were fixed in 10% buffered formalin, at room temperature for 24 h, embedded in paraffin and processed according to routine technique of light microscopy. Sections 5–8 lm thick were stained with hematoxylin and eosin.
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2.3. Protein samples Tissues were homogenized in tissue grinder with RIPA lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA, 0.1% SDS) with 50 mM sodium fluoride and 1 mM sodium orthovanadate, supplemented with 1· Complete Protease Inhibitor (Roche, Indianapolis, IN, USA) and Phosphatase Inhibitor Cocktail I (Sigma–Aldrich, St. Louis, MO, USA). In order to minimize differences in sample preparation procedure, all the samples were processed simultaneously, in standardized conditions. Protein content was measured using Bradford protein assay kit (Bio-Rad, Hercules, CA, USA). Lysates were stored at 80C. 2.4. Western blot Twenty micrograms of protein extracted from tissues were resolved on 12% SDS–PAGE. Afterwards, the gels were transferred onto PVDF membranes. Quality of electrophoresis and transfer was evaluated with Ponceau S staining. Blots were blocked with 5% non-fat dry milk in TBST (Tris buffered saline, 0.05% Tween) and incubated with respective primary and secondary (HRP-conjugated) antibodies. Membranes were washed in TBST buffer and proteins were detected by West Pico chemiluminescence substrate (Pierce, Rocford, IL, USA). 2.5. Immunohistochemistry Immunohistochemical studies were performed on formalin-fixed, paraffin-embedded specimens. Sections of 10 lm were mounted on slides covered with NovoBond (Novocastra, United Kingdom), air-dried for 24 h and deparaffinized through xylene and graded alcohols to PBS (phosphate-buffered saline). To facilitate antibody bounding, the high-temperature antigen unmasking method using citrate buffer was applied. Unspecific bounding sites were blocked with 10% BSA (bovine serum albumin) and 10% goat serum (both from Sigma–Aldrich). Sections were then treated with primary antibodies. In the case of polyclonal antibodies, diluted non-immune serum was used instead of primary antibody, as negative control. For monoclonal antibodies, diluted IgG of relevant isotype was used as negative control. The specimens were kept in humid chamber at 37 C for 1 h. After triple rinsing in PBS, the mixture of secondary antibodies was applied. Staining was performed in moist chamber, at room temperature, for 2 h. Then, after extensive rinsing in PBS, the slides were covered with VectaShield (Vector, USA) and coverslipped. The specimens were examined under the confocal microscope Olympus Fluoview (Olympus, Japan). The pictures of both excitation wavelengths were merged for analysis and data presentation. The semiquantitative analysis
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was done by two independent observers, according to the following criteria: Intensity o staining No signal Weak signal Moderate signal Bright cells
Percent of positive cells 0
0–5
5–25
25–50
>50
+
+ ++
+ ++ +++
++ +++ +++
2.6. Antibodies Antibodies against: b-catenin, phospho-b-catenin (T 33), c-Myc, N-Myc, cyclin D1 and secondary antibodies (HRP-goat anti-rabbit or HRP-bovine anti-mouse) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). For immunohistochemistry, secondary antibodies conjugated with AlexaFluor 488 or AlexaFluor 568 were obtained from Molecular Probes Inc. (Eugene, OR, USA). 2.7. RT-PCR Total RNA was isolated using Trizol reagent (Gibco BRC, Carlsbad, CA, USA) according to manufacturer’s instructions. The RNA pellet was suspended in DEPC water and RNA concentration was assessed by optical density. Two micrograms of RNA was used in reverse transcriptase reaction using oligo(dT)15 primer and M-MLV from Invitrogen (San Diego, CA, USA) according to the protocol of the producer. Expression of GAPDH mRNA was determined by PCR in restricted number of cycles. Twenty cycles of 95 C-3000 , 57 C-3000 and 72 C-3000 with 1 U/25 ll Taq polymerase (Invitrogen), 75 lM of each dNTP and 0.5 lM of each primer raised specific product which was analyzed on 2% TAE agarose gel stained with ethidium bromide. Primers for GAPDH amplifications were: GAPDH forward: 5 0 -AGG TCG GAG TCA ACG GAT TT and GAPDH reverse: 5 0 -CAG CAG AGG GGG CAG AGA TG. After cDNA was normalized to GAPDH, the expression of c-Myc and N-Myc mRNA was evaluated in the similar PCRs. As the efficiency of these reactions was significantly lower, the products visualized in UV were obtained after 30 cycles of 95 C-3000 , 58 C-3000 and 72 C-3000 . Primers for c-Myc, N-Myc and cyclin D1 were as follows: c-Myc forward: 5 0 -ACT CGG TGC AGC CGT ATT TCT ACT, c-Myc reverse: 5 0 -TCT GAC ACT GTC CAA CTT GAC CCT; N-Myc forward: 5 0 -ACA GAC TGT AGC CAT CCG AGG ACA, N-Myc reverse: 5 0 -GAG GTC TGG GTT CTT GCA GAT CAT; cyclin D1 forward: 5 0 -CCC TCG GTG TCC TAC TTC AAA TGT, cyclin D1 reverse: 5’-TGA TCT GTT TGT TCT CCT CCG CCT.
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All primers were synthesized by the Institute of Biochemistry and Biophysics of Polish Academy of Sciences.
3. Results 3.1. SEGAs show typical cell composition with characteristic giant cells Light microscopy showed typical components: spindle-shaped fibrillated cells, polygonal cells with eosinophilic cytoplasm and characteristic giant cells with eccentric vesicular nuclei. Small areas of necrosis (like the one shown in Fig. 1) were seen occasionally.
3.2. b-Catenin levels are increased in SEGAs Western blot of protein extracts from SEGAs demonstrated elevated levels of b-catenin. In SEGA1 and SEGA4 b-catenin was significantly elevated compared to normal human brain, while SEGA2 and SEGA3 showed moderate amount of b-catenin. However, phosphorylation of b-catenin at Ser33, an inevitable step towards its ubiquitination and degradation, was not detected in any of the tested SEGA samples, while it was present in normal brain (Fig. 2). Extracts from human medulloblastoma and hemimegalencephaly, where activation of the WNT pathway is documented [17,18] served as positive controls. In our experiments the presence of b-catenin and no phosphorylation of this protein were detected in both positive control samples.
Fig. 1. Histopathological images of SEGAs. (A, SEGA1) and (B, SEGA2): numerous giant cells with eccentric nuclei; (C, SEGA3): prominent nuclear pleomorphism in a giant cell close to the center of the image; (D, SEGA4): focus of necrosis in the middle of the photo. Bars indicate 50 lm.
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staining in most cells composing the tumor, including typical giant cells. In the majority of cells staining distribution in cytoplasm and nucleus was homogenous (see example in Fig. 3). Control normal human brain samples did not show the presence of b-catenin in the nucleus. 3.4. Detection of protein products of c-Myc, N-Myc and cyclin D1
Fig. 2. Western blot showing reaction with antibodies against WNT pathway-associated proteins. The amount of proteins in lysates was normalized according to anti-actin antibody reaction (not shown). Hemimegalencephaly samples were used as positive control for the activation of the WNT pathway.
3.3. Nuclear translocation of b-catenin Hypophosphorylated b-catenin translocates to the nucleus and fulfils its role as a transcription factor [19]. Fluorescence microscopy demonstrated b-catenin
In order to confirm the activating function of b-catenin, we evaluated by Western blot and immunohistochemistry the expression of three proteins, whose transcription is known to be regulated by the WNT pathway: cyclin D1, c-Myc and N-Myc. Western blot showed strong upregulation of c-Myc in two of four SEGAs (SEGA2 and SEGA3) while in the remaining two cases c-Myc was present at the moderate level. NMyc protein levels corresponded with this result, although the expression of N-Myc was moderate in SEGA2 and SEGA3, and weak in SEGA1 and SEGA4. Surprisingly, cyclin D1 production was not upregulated in any of the SEGAs. The above results were confirmed by immunofluorescent staining (Table 1 and Fig. 3). Presented weakly stained giant cell was an exception in overall negative staining, as evaluated according to our criteria. c-Myc protein was detected at a similar, moderate level in SEGAs 2–4, while it was weak in SEGA1. Also N-Myc was detected, although in this case reaction was significantly weaker. Notably, all three antibodies, including anti-cyclin D1 antibody that showed very weak or no reaction, detected a subpopulation of giant cells with moderate
Fig. 3. Representative immunofluorescent staining of SEGA1 and SEGA2. Original magnification 400·. Arrows show giant cells stained with respective antibodies. Bars link photographs taken under the same visual field. Note homogeneous distribution of b-catenin seen in a giant cell of SEGA2. The same cell demonstrates cytoplasmic staining for N-Myc. Very weak cyclin D1 staining seen in an individual giant cell (see magnification), was classified as negative result, according to our criteria (see Section 2), whereas broader positive staining was detected in SEGA2. Control positive staining with all antibodies (including p-b-catenin and cyclin D1) has been performed with respective normal and tumor tissues, confirming correct detection of these antibodies (results not shown).
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Table 1 Semi-quantitative results of immunohistochemistry of SEGA tissue samples with use of respective antibodies; : no reaction; +: weak reaction; ++: moderate reaction; +++: strong reaction
b-Catenin p-b-catenin (Ser33) Cyclin D1 c-Myc N-Myc
SEGA1
SEGA2
SEGA3
SEGA4
++ +
+ + ++ +
+ + ++ +
+++ + ++ +
Positive control for p-b-catenin (not shown) demonstrated strong cytoplasmic staining.
to strong staining (Fig. 3). Thus, in comparison to normal brain, where cyclin D1 accumulation was not detected, in SEGAs cyclin D1 was only found in a limited number of giant cells. 3.5. Transcription of c-Myc, N-Myc and cyclin D1 Next, we made an attempt to determine whether higher levels of c-Myc protein were accompanied by upregulated transcription. For this purpose, reverse transcription-polymerase chain reaction (RT-PCR) was performed to determine the expression of mRNA for c-Myc, N-Myc and cyclin D1. The reaction showed increased transcription of c-Myc and N-Myc genes in SEGA2 and SEGA3, while the amount of transcripts in SEGA1 and SEGA4 remained on the level of healthy control (Fig. 4). To our surprise, in spite of lack of cyclin D1 protein product, we found its mRNA in all four SEGAs. RT-PCR on positive control (hemimegalencephaly) showed accumulation of mRNA for all three WNT-dependent genes.
Fig. 4. RT-PCR of SEGAs and control samples. Specific bands are shown. cDNAs used for the reaction were normalized according to GAPDH level.
4. Discussion TSC1/TSC2 complex is known to regulate protein translation through its participation in the mTOR pathway. As much as 15% of all proteins in a cell may be regulated by this mechanism [20]. There are, however, numerous age-related differences between different types of TS-associated tumors that cannot be explained by upregulation of mTOR-related translation alone. For example, cardiac rhabdomyomas are detected during embryogenesis and tend to regress after birth, while renal angiomyolipomas (AMLs) develop with age. Also, all three types of TS brain lesions (subependymal nodules, subependymal giant cell astrocytoma and cortical tubers) contain giant cells postulated to result from disturbed cell migration and differentiation. Giant cells have been tested for astrocytic and neuronal markers and found to express them at different levels (for a review see [21]). For these reasons we hypothesized that embryonic growth factors like WNT may contribute to the clinical picture of TS-associated tumors. Positive correlation between TSC2 mutation and amount of b-catenin transcription products has been postulated before. Cyclin D1 mRNA level was demonstrated to be upregulated in giant cells of cortical tubers microdissected from TS patients [22]. It has also been noted that antisense inhibition of tuberin in rat fibroblasts results in increased amount of cyclin D1 protein [23]. Very recently, Mak and collaborators [24] evaluated TS-related AMLs and lymphangioleiomyomatosis (LAM) from the point of view of WNT pathway activation and demonstrated that the tumors contain increased amount of b-catenin-dependent proteins: cyclin D1 and connexin 43. Our evaluation of the WNT pathway products in human brain tumors associated with tuberous sclerosis generally extends the research of Mak and colleagues [24] performed on human AML and LAM sections. There are however some differences in the amount of observed b-catenin-dependent proteins. According to our expectations, c-Myc and N-Myc mRNA was found in two of four SEGAs, and cyclin D1 mRNA was present at variable levels in all four samples. On the protein level however, c-Myc and N-Myc were prominent in the same SEGAs that revealed respective mRNA transcripts, while cyclin D1 did not appear in Western blot, and in situ immunostaining showed very scant reaction. It is worth stressing that all three antibodies against b-catenin-dependent proteins demonstrated positive staining in a population of giant cells, typical for the development of SEGA. Histological composition of both types of tumors, AML and SEGA, may contribute to our understanding of the differences in cyclin D1 presence and/or distribution. While AMLs are composed of varying amounts of thick-walled blood vessels, mature adipose tissue and smooth muscles [25], SEGAs
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contain three types of cells: astrocytes, dysmorphic neurons and characteristic giant cells [26]. Differential expression of cell cycle-related cyclin D1 in both types of lesions may also reflect the rate of their development: SEGAs are generally slowly growing neoplasms [27], while AMLs have a tendency to develop more rapidly [28]. One more explanation of the discrepancy in the amount of cyclin D1 mRNA and protein seems to be tempting. In 2004 Koziczak and Hynes [29] evaluated the cooperation of ErbB2 and fibroblast growth factor receptor-4 (FGFR-4) in the regulation of cyclin D1 production. The authors found that FGFR-4, triggering the Erk pathway, is responsible for the control over cyclin D1 transcription, while ErbB2, acting through Akt, is responsible for the increase in cyclin D1 translation. In our recent paper [30], performed on the same four SEGA samples, we have detected strong upregulation of the Erk pathway in all four SEGAs, and weak upregulation of Akt only in SEGAs 3 and 4. Thus, high levels of cyclin D1 mRNA in all tested SEGAs could result from high Erk activity, while low amount of the protein may be explained by weak Akt phosphorylation. Mak and colleagues [24] determined that the distribution of b-catenin staining within the tumors was variable and that b-catenin activation may be limited to the outer limits of the tumor. SEGA samples tested in this study were not collected from the outer part of tumors, which may additionally explain discrepancy with the results of Mak. It is noteworthy, that immunohistochemistry revealed nuclear staining of b-catenin in our samples. According to Eberhart and colleagues [19], active form of b-catenin implies activation of the WNT signaling pathway. The authors examined 48 glial and meningeal CNS tumors, all of which were negative for nuclear b-catenin, finding this type of reaction only in medulloblastoma samples, where participation of the WNT pathway is well-documented. Our results demonstrate that development of SEGA is accompanied by the activation of the WNT pathway, leading to transcription and overproduction of cell cycle-regulating proteins like c-Myc or N-Myc. However, precise mechanism and significance of this finding requires further research.
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