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Keywords:

  • brain tumors;
  • glioma;
  • Sox2;
  • Sox21;
  • S100

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Sox2 is a transcription factor in neural stem cells and keeps the cells immature and proliferative. Sox2 is expressed in primary human glioma such as glioblastoma multiforme (GBM), primary glioma cells and glioma cell lines and is implicated in signaling pathways in glioma connected to malignancy. Sox21, the counteracting partner of Sox2, has the same expression pattern as Sox2 in glioma but in general induces opposite effects. In this study, Sox21 was overexpressed by using a tetracycline-regulated expression system (tet-on) in glioma cells. The glioma cells were injected subcutaneously into immunodeficient mice. The control tumors were highly proliferative, contained microvascular proliferation and large necrotic areas typical of human GBM. Induction of Sox21 in the tumor cells resulted in a significant smaller tumor size, and the effect correlated with the onset of treatment, where earlier treatment gave smaller tumors. Mice injected with glioma cells orthotopically into the brain survived significantly longer when Sox21 expression was induced. Tumors originating from glioma cells with an induced expression of Sox21 exhibited an increased formation of Sox2:Sox21 complexes and an upregulation of S100β, CNPase and Tuj1. Sox21 appears to decrease the stem-like cell properties of the tumor cells and initiate aberrant differentiation of glioma cells in vivo. Taken together our results indicate that Sox21 can function as a tumor suppressor during gliomagenesis mediated by a shift in the balance between Sox2 and Sox21. The wide distribution of Sox2 and Sox21 in GBM makes the Sox2/Sox21 axis a very interesting target for novel therapy of gliomas.

Glioblastoma multiforme (GBM) is the most frequent primary brain tumor and the most malignant neoplasm in the central nervous system (CNS) with a mean survival time of ∼1 year.[1, 2] Recent studies suggest that initiation and progression of gliomas are driven by brain tumor initiating cells (BTICs).[1, 3-5] BTICs have the ability to regrow the tumor and sustain tumor growth through their self-renewal potential. BTICs have stem-like properties in the sense that they are multipotent and can be induced to express markers of different cell lineages.

Sox (Sry-related HMG box) genes encode transcription factors which regulate CNS development and other developmental processes.[6, 7] Sox2 and Sox21 belong to the B1 and B2 subgroups, respectively, and are often coexpressed during development. They are assumed to target the same genes but with opposite effects, since Sox2 contains an activating domain and Sox21 a repressing domain.[8, 9] Targeted genes are suggested to be regulated positively or negatively depending on the balance between the two transcription factors.[10]

Postnatal neural stem cells are located in the subventricular zone and in the hippocampus dentate gurys, and these cells are positive for Nestin, GFAP as well as Sox2.[11, 12] Downregulation of Sox2 with siRNA blocks the proliferation of neural stem-like cells and induces neuronal differentiation.[13] Similarly, neurogenesis can be blocked in the chick neural tube by overexpression of Sox2 together with other B1 group members, Sox1 and Sox3.[14] However, forced expression of Sox21 in the chicken neural tube promotes neurogenesis by counteracting the activity of Sox1–3. Thus, the balance between Sox1–3 and Sox21 seems to be an important deterministic factor for neural cells to either remain in a progenitor state or to be committed to differentiation.[15] Further, ectopic expression of Sox21 in embryonic stem (ES) cells interrupts the pluripotency and the self-renewal capacity of ES cells and induces neuroectodermal differentiation.[16]

In GBM, Sox2 is amplified in about 9% and overexpressed in 86% of the tumors, which suggests that Sox2 can act as an oncogene in glioma.[17] Sox2 is identified as an oncogene in other tumor types such as lung cancer,[18] breast cancer[19] and colorectal cancer.[20] We have recently shown that Sox21, like Sox2, is expressed in both adult and childhood brain tumors such as GBM, astrocytoma, oligodendroglioma, medulloblastoma, primitive neuroectodermal tumors and ependymoma.[9] Upregulation of Sox21 in glioma cells resulted in decreased expression of Sox2 and GFAP and further, forced expression of Sox21 induced apoptosis and resulted in impaired proliferation of glioma cells in vitro. The same results were observed when Sox2 was knocked down with siRNA.[9]

The aim of this investigation was to elucidate further the function of Sox21 in gliomas in vivo. For these experiments, we used the human U-343 MG-a glioma cell line, which has an endogenous expression of Sox2, Sox21 and GFAP similar to GBM in vivo. Further, the U-343 MG-a cell line is clonable and tumorigenic in mice[9, 21] and thus a valuable tool for studies both in vivo and in vitro. We used a tetracycline inducible cell system (tet-on) to upregulate Sox21 expression in glioma cells.[9] Human glioma-Sox21-tet-on cells were injected subcutaneously into the flank or orthotopically in immunodeficient mice, which were treated with tetracycline and doxycycline, respectively, to induce Sox21. Expression of ectopic Sox21 in glioma cells in vivo resulted in a significant reduction of tumor size and prolonged survival, further several differentiation factors, namely, S100β, CNPase and Tuj1, were upregulated suggesting that increased expression of Sox21 inhibits proliferation and induces differentiation.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Expression vectors and cell clones

We have previously generated stable human glioma cell clones[9] where a Sox21-myc cDNA construct was cloned into a tetracycline-regulated system (T-Rex System, Invitrogen, Carlsbad, CA). Four stable cell clones were used: clone A1-Sox21, A2-Sox21 and A3-Sox21, which all are Sox21 inducible clones. Clone B1-empty is a negative control clone containing the vectors but without Sox21-myc cDNA.[9]

Antibodies

Primary antibodies: polyclonal anti-Sox2 (AB5603; Millipore, Temecula, CA), polyclonal anti-Sox2 (AF2018; R&D systems, Minneapolis, MN), polyclonal anti-Sox21 (GT15209; Neuromics, Edina, MN), monoclonal anti-c-myc (9E19; Santa Cruz, Heidelberg, Germany), polyclonal anti-GFAP (Z0334; Dako, Glostrup, Denmark), polyclonal anti-GFAPδ (ab28926; Abcam, Cambridge, United Kingdom), polyclonal anti-Nestin (AB5922; Millipore), monoclonal anti-CNPase (C5922; Sigma, Saint Lous, Missouri), monoclonal anti-Tuj1 (MMS-435P; Covance), polyclonal anti-cleaved caspase-3 (#9661; Cell Signaling, Danvers, MA), polyclonal anti-phospho Histone H3 (pH3; #06–570 Millipore) and polyclonal anti-S100 (Z0311; Dako). Anti-S100 binds strongly to S100β on western blots, while binding to A1 and A2 isoforms are weak and very weak, respectively. Reactivity to other tested isoforms, (A2, A3 and A4) is absent (data sheet of Z0311; Dako). Peroxidase-labeled secondary antibody for western blots; anti-mouse IgG, anti-rabbit IgG (GE Healthcare United Kingdom) and anti-goat IgG (Dako). Secondary antibodies for immunofluorescence; Alexa Flour 555 donkey-anti rabbit, Alexa Flour 488 donkey anti-rabbit and Alexa Flour 555 donkey anti-goat (Invitrogen). Secondary antibody for immunohistochemistry; biotinylated immunoglobulin rabbit anti-goat (Dako).

Sphere culture

The cell clones were cultured as spheres as previously described[22] with the addition of 1.5 μg/ml Blasticidin and 600 μg/ml Zeocin (Invitrogen). Sox21-myc was induced by treating the cells with tetracycline daily at a concentration of 1 µg/ml, and control cells were treated with absolute ethanol. Sphere cultures were incubated for a minimum of 14 days. Pictures were taken using an Olympus microscope (Olympus Optical, Japan).

Cell cultures established from subcutaneous tumors

Cell cultures were established in stem cell medium from whole tumor biopsies by mechanical mincing and incubation in trypsin for 15 min. The dispersed tumor tissue was cultured until adherent cells could be visualized in the petri dish, about one and a half month and subsequently cultured as described.[9] Sox21-myc was induced with tetracycline as for the spheres. Cultures were established from tumors of clone A2-Sox21 and B1-empty treated with sucrose or tetracycline from the day of injection.

Human glioblastoma stem cell cultures

New human glioblastoma stem cell cultures were established from fresh tumor samples from adult patients.[23] After primary sphere formation cells were cultured as adherent cells on primaria tissue culture dishes (Falcon) coated with laminin (Sigma).[24] The human glioblastoma stem cell cultures, U-3005 MG, U-3013 MG, U-3024 MG, U-3031 MG and U-3047 MG, were used at passage 12–41. Cells were fixed with 4% PFA for 10 min prior to immune staining.

Subcutaneous tumor induction

All animal experiments were performed in accordance with the local animal ethics committee. CB-17 SCID mice were purchased from Taconic, Denmark. Adult mice were injected subcutaneously at the age 4–6 weeks in the left flank with 200 μl PBS and 5 × 106, 8 × 106 or 1 × 107 cells for determination of tumor take. In all other experiments, 8 × 106 cells were used for injection. Mice were treated with 2.5% sucrose or a mixture of 2.5% sucrose and 5 mg/ml tetracycline in drinking water throughout the experiments. A third group of mice received the mixture 4.5 weeks after injection. Mice were euthanized after 12 weeks. The tumor volume was calculated using the ellipsoid formula (V = L × W × H × π/6). The tumors were fixed in 4% formalin and embedded in paraffin.

Orthotopic tumor induction in the brain

Neonatal mice (P1–3) were injected in the right cerebral hemisphere with 4 µl containing 1 × 105 or 8 × 105 clone A2-Sox21 or B1-empty cells. One group of mice received doxycycline-containing food (625 mg doxycycline/kg, Harlan Laboratories, Netherlands) while the other group received regular food. Mice were monitored regularly and euthanized when presenting with symptoms, 12 or 24 weeks after injection. Brains were embedded in paraffin, sectioned and analyzed after hematoxylin and eosin (H&E) stainings. Pictures were taken using a Leica DMRE microscope (Leica Microsystems, Wetzlar, Germany).

EdU injections and detection

For 5-ethynyl-2′-deoxyuridine (EdU) injections each mouse was given a total amount of 200 μg of EdU in a volume of 100 μl PBS through intraperitoneal (IP) injection at 6, 22 and 28 hr prior to euthanization. EdU-stainings were performed with Click-iT EdU Alexa Flour 488 imaging kit according to the manufacturer's protocol (Invitrogen).

Immunohistochemistry and immunofluorescence

Immunochemical and immunofluorescence stainings were performed on 6 μm sections or cell cultures with different primary antibodies.[9] For immunohistochemistry, the UltraVision LP detection system and DAB substrate kit was used according to the manufacturer's protocol (Thermo Scientific, Cheshire, United Kingdom). For immunohistochemistry with goat antibodies, the Vectastain ABC system and DAB substrate kit was used (Thermo Scientific).

In situ proximity ligation assay (in situ PLA)

In situ PLA was performed on 6 µm sections of subcutaneously injected tumors using the Duolink In Situ kit (Olink Bioscience, Uppsala, Sweden). Duolink II PLA probes were used as follows; for detection of Sox2 and the Sox2:Sox21 complex, double recognition, anti-Rabbit PLUS and anti-Goat MINUS, for detection of Sox21 anti-Goat PLUS and MINUS. The slides were counter stained with DAPI.

Western blots

Western blot analysis was performed as described previously.[9] Protein extractions were performed after 14 days in the presence of tetracycline or ethanol as described.

Image analysis

Unless otherwise stated, pictures were taken with a Zeiss Axio Imager.M2 microscope (Carl Zeiss Micro imaging Gmbh, Gottingen, Germany). To quantify EdU, pH3, c-myc and cleaved caspase-3 expression, the fraction between positive cells and total number of cells was determined. In situ PLA quantification was done by calculating the number of signals per area unit stained with DAPI. For Tuj1, CNPase and S100, a ratio between the intensely stained cytoplasmic area and the total cytoplasmic area was calculated. All image analysis was done with CellProfiler software.[25]

Statistical analysis

To analyze subcutaneous tumor sizes, 1-way ANOVA with Dunn's multiple comparison test was used. For mice with orthotopically induced tumors, a log-rank test was performed and Kaplan-Meier curves plotted. For all other analyses, two-tailed Mann-Whitney tests were done. All statistical analysis was performed using the GraphPad Prism 5 software.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Sox2 deficient cells do not form proper spheres and upregulation of Sox21 delays sphere formation in Sox2 expressing cells

Recently, we showed that ectopic expression of Sox21 in Sox2+ glioma cells reduced the expression of Sox2, decreased cell proliferation and induced apoptosis in vitro.[9] In this study, we investigated if the Sox21 inducible glioma clones are tumorigenic and if the tumor growth is affected by Sox21 induction. We used a tetracycline-inducible cell system where the expression of Sox21 can be effectively upregulated. Three inducible glioma-Sox21 tet-on clones were used: clone A1-Sox21, clone A2-Sox21, clone A3-Sox21 and the control clone B1-empty.[9] We started to investigate if the Sox21 inducible clones were able to form spheres in culture; the ability to form spheres expressing several lineage markers is a commonly used criterion of BTICs.[26, 27] Three out of four clones efficiently formed spheres; clone A1-Sox21, clone A2-Sox21 and the control clone (Figs. 1a1d). Induction of Sox21 by tetracycline in the clone A1-Sox21 and clone A2-Sox21 delayed the sphere formation (Figs. 1a and 1b), whereas the sphere forming ability of the B1-empty clone was unaffected (Fig. 1d). Clone A3-Sox21, with low endogenous Sox2 expression[9] formed aggregates rather than spheres and there was no significant difference between treatments (Fig. 1c). Immunohistochemistry staining of Sox21 showed a significant upregulation of Sox21 in tetracycline-treated spheres compared with the control clone (Supporting Information Fig. 1). These results indicate that induced expression of Sox21 delays sphere formation. To test the tumorigenic ability of the clones, cells were injected subcutaneously into the flank of SCID mice. Only the Sox2 positive clones, that is, clone A1-Sox21, clone A2-Sox21 and the control clone B1-empty, formed tumors (Fig. 1e). Clone B1-empty proliferated fastest and gave rise to largest tumors. Mice injected with the Sox2 negative clone A3-Sox21 did not develop tumors regardless of the injected number of cells (Fig. 1e). The results imply that downregulation of Sox2 through induced expression of Sox21 or endogenous lack of Sox2 causes reduced tumor-initiating capacity.

image

Figure 1. The Sox2 deficient clone does not form proper spheres, the upregulation of Sox21 induces delayed sphere formation in Sox2 expressing cells and the sphere forming capacity is reflected in the tumor initiating capacity. Sphere formation assay of the Sox21 inducible clone A1-Sox21 (a), clone A2-Sox21 (b), the Sox21 inducible but Sox2 deficient clone A3-Sox21 (c) and the control clone B1-empty (d) treated with ethanol or tetracycline as indicated. Pictures of the sphere cultures were taken at day 7, the experiments were ended after 14–21 days of incubation with no further differences. Upregulation of Sox21 resulted in delayed sphere formation. Scale bar 100 µm (n = 3). The sphere forming ability reflected the tumor initiating capacity. (e) Tumor cells from each clone, respectively, were injected subcutaneously into the flank of SCID mice. Injected number of cells and tumor volume is indicated in respective diagram. The control clone B1-empty formed spheres faster and formed the largest tumors. The Sox2 deficient clone A3-Sox21 did not have any tumorigenic capacity (n = 3). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Sox21 inhibits progression of subcutaneous tumors

We continued to investigate the histology of the subcutaneous tumors. Uninduced tumors displayed microvascular proliferation, an abundance of mitotic cells and large necrotic areas, occasionally with a tendency to form perinecrotic palisading cells (Fig. 2d), all typical signs of GBM. Mice injected with the inducible clone A2-Sox21 and treated with tetracycline at the day of injection developed tumors that resembled GBMs, but the tumors were significantly smaller than those detected in sucrose treated animals (Figs. 2a and 2c). Mice treated with tetracycline 4.5 weeks after transplantation developed slightly larger tumors compared to mice, which received tetracycline at the day of injection but evidently smaller than sucrose treated tumors (Fig. 2a). Further, tumors developed in mice treated with tetracycline at the day of injection rarely contained necrotic areas while mice treated with tetracycline after 4.5 weeks developed tumors that resembled those occurring in the nontreated mice but with significantly fewer and smaller necrotic areas. This result was also seen for the second Sox2+ clone A1-Sox21 (not shown). Mice injected with the control clone, developed tumors faster and the tumors were larger compared to the inducible clone A2-Sox21 and there was no difference in tumor size between treatments (Fig. 2).

image

Figure 2. The tumor size is significantly smaller in subcutaneous tumors with induced expression of Sox21 and the effect is more pronounced after early onset of treatment. Subcutaneous tumor size distribution for (a) the Sox21 inducible clone A2-Sox21 and (b) clone B1-empty when treated with sucrose (untreated), tetracycline from the day of injection (Tet-S) and tetracycline after 4.5 weeks (Tet-A). Treated tumors were significantly smaller and the effect was more prominent at earlier treatment. 1-way ANOVA with Dunn's multiple comparison test was used for the statistical analysis of tumor size, outliers are depicted in red. p** <0.01, p*** <0.001 (n = 10 and 13), respectively. Number of injected cells: 8 × 106. (c) Representative pictures show hematoxylin and eosin (H&E) stainings of sucrose and tetracycline treated tumors as indicated, 12 weeks after injection and treatment. Scale bar 1000 µm. (d) H&E stainings of representative untreated tumors showing from left; tumor containing a large necrotic area, tumor cells resembling perinecrotic palisading cells and microvessels. These features were present in all tumors regardless of clone or treatment. Treated clone A2-Sox21 had small if any necrotic areas. Scale bar 50 µm.

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Tumors originating from the Sox21 inducible clone showed an obvious but heterogeneous upregulation of Sox21 in tetracycline-treated mice compared with control treated (Supporting Information Figs. 2A and 2B). Highest expression of Sox21 was seen in cells close to the necrotic areas (when present) and the expression of Sox21 was gradually reduced from the necrotic areas and outwards (Supporting Information Figs. 2E and 2F). Our data indicate that the tumor growth can be significantly retarded by induction of Sox21.

Induced expression of Sox21 increase complex binding of Sox2

Next, we examined if Sox2 and Sox21 physically interact in the tumors. We used the in situ PLA method[28] to quantify the expression of Sox2 and Sox21 and to detect possible protein-protein interaction in the subcutaneously induced tumors. As expected, there was a significant increase of Sox21 in the tetracycline treated tumors compared with the sucrose treated tumors (Fig. 3a). Analysis of the expression of Sox2 showed the same expression levels in sucrose treated tumors as in tetracycline treated tumors (Fig. 3b). Double recognition analysis using one antibody against Sox2 and one against Sox21 showed that Sox2 and Sox21 form a protein complex with a significant increase of the Sox2:Sox21 complex after induction of Sox21 (Fig. 3c). Thus, increased expression of Sox21 changes the balance and composition between Sox2 and Sox21 in treated tumors. This finding is in line with the notion that modified expression of Sox21 changes the actions of Sox2 here by complex formation.

image

Figure 3. Induced expression of Sox21 results in an increased amount of Sox2 bound to a Sox2:Sox21 complex. Each panel shows subcutaneous tumors of Clone A2-Sox2, derived from mice treated with sucrose (untreated) and tetracycline from the day of injection (Tet-S). Representative pictures show the in situ PLA staining for (a) Sox21, (b) Sox2 and (c) complex bound Sox2 and Sox21. The graphs to the right show the quantification of the number of signals per area unit in the nucleus. A two tailed Mann-Whitney test was used for statistical analysis. p**<0.01. n = 4−6. Scale bar 40 µm.

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Induced expression of Sox21 stimulates aberrant differentiation of glioma cells and provokes apoptosis

Given that Sox21 promotes neuronal differentiation in neural progenitor cells by counteracting Sox1–3[15] we studied the expression of different cell lineage markers in the subcutaneous tumors. All tumors, regardless of origin or treatment, were strongly positive for Nestin, GFAP as well as GFAPδ, indicating that the tumor cells have astrocytic and/or progenitor properties (Supporting Information Fig. 3A and 3B). Tumors originating from clone A2-Sox21 treated with tetracycline showed a remarkable increase in S100β expression and signs of increased CNPase expression compared to untreated tumors (Supporting Information Fig. 3C–3E). No significant difference in Tuj1 expression was detected, although the three differentiation markers showed an overlapping expression pattern (Supporting Information Fig. 3C–3E). S100β was not expressed in tumors from the clone B1-empty regardless of treatment (not shown). Our results indicate that ectopic expression of Sox21 induces glioma differentiation.

We have previously reported that overexpression of Sox21 induces apoptosis in vitro,[9] and here investigated if this phenomenon also occurred in vivo. Tumors originating from clone A2-Sox21 treated with sucrose or tetracycline were both positive for cleaved caspase-3 around necrotic areas. However, outside of these areas there was a significant increase of cells positive for cleaved caspase-3 in tetracycline-treated tumors (Supporting Information Fig. 4). Upregulation of Sox21 thus provokes apoptosis in glioma cells in vivo.

Sox21-induced differentiation is sustained and cells from nontreated tumors maintain the capacity to upregulate Sox21

The stability of the phenotype induced by Sox21 overexpression was analyzed in cell culture. Twelve weeks after subcutaneous transplantation, cell cultures were established from tumors originating from clone A2-Sox21 and clone B1-empty. Explanted cells were cultured and expanded for 4–6 weeks in tetracycline-free medium and thereafter used for experiments. Both ectopically induced Sox21 and the total amount of Sox21 were upregulated in tetracycline treated cells from tumors of clone A2-Sox21, in cells originating from tumors treated with tetracycline, as well as in cells from untreated tumors (Fig. 4a). There were no changes in expression of Sox21 or Sox2 in the control clone regardless of treatment. However, after 12 weeks of tumor growth in vivo, the inducible tumor cells, irrespectively of tumor origin, still responded to tetracycline treatment by an upregulation of Sox21 in vitro. We continued to investigate the expression of Sox2 in the explanted cells. After induction of Sox21, a clear decrease in Sox2 expression was seen in cells established from the clone A2-Sox21 tumor treated with tetracycline as well as in tetracycline-treated cells from the sucrose treated tumor (Fig. 4a). Tumor cells from tetracycline-treated tumors grown in tetracycline-free medium regained their Sox2 expression and had an increased endogenous expression of Sox21, indicating that the effect of Sox21 on Sox2 expression is reversible (Fig. 4a).

image

Figure 4. Cell cultures established from tumors originating from the A2-Sox21 clone continued to express S100β and downregulated progenitor markers upon induction of Sox21. Western blot analysis of cells cultured in vitro derived from clone B1-empty and clone A2-Sox21 tumors treated with sucrose (untreated) or tetracycline in vivo for 12 weeks. After establishment of the cell cultures, the cells were cultured in the presence of ethanol (EtOH) or tetracycline (Tet). Protein extractions were performed after day 14 of treatment. (a) Forced expression of Sox21 was detected with antibodies against Sox21 and c-myc (detecting Myc tagged Sox21). The total amount of Sox21 is increased in tetracycline treated cells originating from the clone A2-Sox21 tumor, regardless of if the tumor was treated with sucrose or tetracycline. Fourteen days of induction of Sox21 resulted in decreased Sox2 expression. (b) GFAP and GFAPδ are both reduced in tetracycline treated cells. (c) The expression of Tuj1 and CNPase was unaffected; S100β was upregulated in tetracycline treated cells. The upregulation of S100β expression in vivo persisted in vitro even in cells, which did not received tetracycline, supporting a long-term differentiation effect of Sox21 (n = 2). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Cultured cells from the tumor of clone A2-Sox21 expressed both GFAP and GFAPδ and both markers were reduced after upregulation of Sox21, most evident in cells originating from the tumors treated with tetracycline due to the long-term exposure (Fig. 4b). Cells from the control clone did not show any changes in GFAP or GFAPδ expression. Neither did the expression of the neuronal marker Tuj1 or the oligodendrocytic marker CNPase differ between tumor origin and treatment (Fig. 4c). However, there was a striking upregulation of the expression of S100β in cells from the Sox21 inducible clone A2-Sox21 treated with tetracycline (Fig. 4c), consistent with the in vivo findings (Supporting Information Fig. 3). Thus, the effect of Sox21 on S100β expression is at least partially sustained in tetracycline-free medium.

Increased expression of Sox21 prolongs survival by inducing differentiation

We continued to investigate how an increased expression of Sox21 affects glioma cells after orthotopic transplantation in mice. The clone A2-Sox21 and the control clone were injected intracerebrally in neonatal mice. Mice were fed with food with or without doxycycline from the day of injection and sacrificed when presenting symptoms or after 12 or 24 weeks. Mice injected with the inducible clone survived significantly longer upon doxycycline treatment compared to untreated mice (Fig. 5a). All mice injected with the inducible clone survived to the 12 week endpoint while treated, but all exhibited small tumors (Figs. 6a and 6b). In the untreated group, none of the mice survived the 12 week period, the mean survival time was 52 days (7.4 weeks). H&E staining revealed that untreated mice developed relatively small tumors but treated mice presented even smaller tumors after 12 weeks incubation (Figs. 6a and 6b). In the 24 week experimental group, a few large tumors appeared in treated mice. Surprisingly, the treated mice appeared to cope with the tumor better than the untreated mice (Figs. 6c and 6d). Mice with induced expression of Sox21 in the 24 week experimental group had a mean survival of 144 days compared to 52 days for untreated mice (Fig. 5b). Mice injected with the control clone did not respond to the doxycycline-treatment, the mice became ill earlier and the tumors appeared more aggressive. The mean survival of the control clone was 32 and 29 days, respectively, for treated and nontreated mice (Fig. 5).

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Figure 5. The survival increases significantly with induced expression of Sox21 in the glioma cells. (a) Kaplan-Meier analysis show the survival of mice injected with the Sox21 inducible clone A2-Sox21 or the control clone B1-empty as indicated. Neonatal mice were injected with 8 × 105 cells into the brain. Mice were feed with doxycycline containing food (Dox-treated) or with regular food (untreated) from time of injection. Mice were sacrificed when ill or 24 weeks after injection. Log-rank (Mantel-Cox) test was performed for the Kaplan-Meier analyses and showed significant differences in survival between clone A2-Sox21 Dox-treated and clone A2-Sox21 untreated. (b) The table indicates percentage of mice that developed tumors and mean survival time in days. p****<0.0001 (n = number of injected mice in each group).

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image

Figure 6. Induced expression of Sox21 results in a higher expression of Sox21 and an induced expression of differentiation markers. (a–e) Representative pictures showing from left H&E stainings, immunohistochemical stainings for Sox21 (total amount), Sox21-Myc-tagged (induced Sox21) and Sox2 from tumors derived from mice injected with the clone A2-Sox21 feed with doxycycline containing pellets (Dox-treated) or regular pellets (untreated) from the day of injection. (a) Shows a tumor from an untreated mouse, sacrificed after 6.4 weeks of incubation due to symptom of illness (mouse belonging to the 12 week group). (b) Tumor from a doxycycline treated mouse, sacrificed after 12 weeks of incubation. (c) Tumor from an untreated mouse, sacrificed after 6.4 weeks of incubation due to symptom of illness (mouse belonging to the 24 week group). Tumors from mice treated with doxycycline, sacrificed after 20 weeks incubation because of symptom of disease (d), respectively, after 24 weeks of incubation, the end point of experiment (e). Scale bar 100 and 500 µm, respectively, for H&E and 50 µm for the immunohistochemical stainings. (f, g) Representative pictures showing immunohistochemical stainings for Tuj1, CNPase and S100β as indicated. (f) Mouse injected with clone A2-Sox21 feed with regular pellets (untreated) from day of injection sacrificed at 10.7 weeks, and (g) mouse feed with doxycycline-containing pellets (Dox-treated) from the day of injection, sacrificed at 17.6 weeks. Scale bar 30 µm. (h) Quantification of Tuj1, CNPase and S100β expression. The graphs show the ratio between the intensely stained cytoplasmic area and the total cytoplasmic area. A two-tailed Mann-Whitney test was used for statistical analysis. p*< 0.05, p**< 0.01 (n = 4−6).

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Immunohistochemical analysis revealed an upregulation of Sox21 in the doxycycline treated tumors originating from the Sox21 inducible clone, as shown both by Sox21 and myc-tag stainings (Figs. 6a6e). Sox2 was, as with the subcutaneous tumors, still expressed in the tumors (Figs. 6a6e). Doxycycline treatment had no effect on Sox2 or Sox21 in tumors originating from the control clone (not shown). In conclusion, induced expression of Sox21 in glioma significantly increases the survival time and delays the progression of the tumors.

Treated tumors showed a significant upregulation of Tuj1, CNPase and S100β (Figs. 6f6h) compared with untreated tumors. The expression of Nestin, GFAP and GFAPδ was unaffected by doxycycline treatment (Supporting Information Fig. 5). This suggests that induced expression of Sox21 at least in part delays glioma progression through differentiation.

Induced Sox21 expression reduces glioma cell proliferation

We continued to examine if Sox21 had an effect on tumor cell proliferation in vivo. We have earlier shown that induced expression of Sox21 has a modest effect on cell proliferation in vitro.[9] Mice injected orthotopically with clone A2-Sox21 cells were injected intraperitoneally with EdU prior to euthanization at the experimental endpoint. Although not significant, the number of EdU positive cells was reduced in doxycycline-treated tumors (Supporting Information Fig. 6A). We continued to investigate the cell proliferation of the orthotopically injected tumors of clone A2-Sox21 by staining for the mitosis marker phospho-Histone H3 (pH3).[29] Quantification of pH3 positive cells also showed a tendency of reduced proliferation rate in the tumors after 12 weeks of doxycycline treatment (Supporting Information Fig. 6B). These results indicate that ectopic expression of Sox21 leads to a restricted proliferative capacity, which may result in increased survival time.

Sox2 and Sox21 are coexpressed in human glioblastoma

Previously, we have shown that mRNA expression levels of Sox2 and Sox21 are positively correlated in glioma.[9] This correlation was confirmed when analyzing the data set from the Cancer Genome Atlas (TCGA; not shown).[30] To verify the expression of Sox2 and Sox21 in GBM, we stained five newly isolated GBM patient-derived human glioblastoma stem cell cultures for Sox2 and Sox21 expression. All five human glioblastoma stem cell lines stained positive for both Sox2 and Sox21 and the expression was overlapping in all cell lines irrespectively of passage number (Supporting Information Fig. 7). These results confirm that there is a correlation between Sox2 and Sox21 in glioma. Further, the cell lines also stained positive for GFAP in line with previous studies (not shown).[9, 31, 32]

Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Recent data suggest that initiation and progression of brain tumors are driven by cancer stem-like cells[26, 27] and several studies have shown that Sox2 is important for keeping normal neural stem cells in a proliferating and immature state.[11-13] Sox2 is widely expressed in glioblastoma as well as in several glioma cell lines,[9, 33-35] although the major proportion of cell lines established in serum-containing medium are Sox2 negative, which is not the case when established in defined stem cell medium.[9, 31, 32]

A functional role for Sox2 in gliomagenesis has previously been demonstrated. Thus, knock down of Sox2 by microRNA blocks the tumor initiating capacity of GBM cells after orthotopical injection in NOD/SCID mice[36] and downregulation of Sox2 by siRNA in BTICs results in loss of self-renewal capacity.[17] Similarly Ge et al. showed that overexpression of Sox2 in glioma cells causes an increased number of neurospheres suggesting that Sox2 is involved in self-renewal of glioma.[37] In line with these reports, we found that only Sox2 positive glioma clones gave rise to tumors with extensive necrosis and highly proliferating cells. Clone A3-Sox21 with no or only low Sox2 levels[9] did not yield any tumors, further demonstrating that Sox2 deficiency results in an inability to form tumors. In contrast to the Sox2 expressing clones, this clone was not able to form proper spheres. In addition, we show that newly established patient-derived stem cell cultures from GBM express both Sox2 and Sox21.

While a few reports suggest that Sox21 can promote differentiation of neural stem cells by counteracting the activity of Sox2,[15, 35] there are surprisingly few reports about the role of the Sox2/Sox21 axis during brain tumor development. Mallana et al. have shown that Sox2 and Sox21 can be coimmunoprecipitated in ES cells[16]; here, we show for the first time that Sox2 and Sox21 form a protein complex in vivo in glioma and that induced expression of Sox21 increases the number of interactions, which changes the balance towards more Sox2:Sox21 complexes (Fig. 3). This investigation provides evidence that tumor growth is inhibited by Sox21 overexpression, probably due to the enhanced amount of protein complexes. An increase of the expression of Sox21 by transducing an inducible vector significantly reduced the tumor size and increased the survival of xenotransplanted mice (Figs. 2 and 5). However, Sox21 did not block tumorigenesis completely under these conditions, which is in line with delayed sphere formation of Sox2 positive cells after induction of Sox21. We have previously shown that human gliomas may have a relatively high expression of Sox21,[9] which may seem paradoxical given the notion that Sox21 is a tumor inhibitory factor. However, high expression of Sox21 is only seen in tumors with high expression of Sox2 and here, we show that all tested freshly established GBM cell lines co-express Sox2 and Sox21. We have also shown that the expression of Sox2 and Sox21 co-vary in GBM both within the IST[9] and the TCGA database (data not shown). We also found that the expression of Sox2 or Sox21 is not associated with survival, in line with the findings of Wan et al., who showed that the expression of Sox2 is not correlated to survival.[38] Our working hypothesis is that the absolute levels of Sox2 or Sox21 are not as important as the ratio between the two factors. It is therefore interesting to note that the analysis of Sox2/Sox21 complexes by PLA indicated that the level of unbound Sox2 decreased upon Sox21 induction. In addition, Holmberg et al. has demonstrated that the co-expression of Sox2 and neural stem cell markers such as Oct4 increases with malignancy in glioma.[39]

In mouse embryonic fibroblasts induced expression of Sox2 induces Sox21 and induction of Sox21 by Sox2 is necessary to induce the pluripotent stem cell state.[40] It has also been shown that Sox2 can bind the Sox21 enhancer in ES cells.[40, 41] Possibly, it is the balance between Sox2 and Sox21 or preferably the Sox2:Sox21 interactions, rather than the absolute levels of Sox21, which is decisive. This is supported by our in vitro cultures; when the induction of Sox21 is withdrawn this threshold is no longer reached and compensatory mechanisms upregulate Sox2 and to some extent the endogenous expression of Sox21. Indeed, Mallanna et al. showed that upon induction of Sox2, Sox21 is upregulated, which results in differentiation in ES cells, whereas induction of Sox21 inhibits Sox2 expression in the cells.[16] On the contrary, Cox et al. showed that a 2–3 fold increase of Sox2 levels in glioblastoma and medulloblastoma cells extensively impairs their capability to proliferate and form spheres.[42] Together, these results suggest that the expression of Sox2 must be precisely controlled by the tumor cells during gliomagenesis. Shifting the Sox2/Sox21 balance by transduction of a Sox21 expression vector, may therefore lead to an inhibition of the tumor promoting activity of Sox2, as we observed in this investigation.

The reduced tumor size and the increased survival of the mice results from induced Sox21 expression, which was accompanied by an abnormal differentiation of the cells with a concomitant expression of several lineages markers. Mallana et al. have shown that induction of Sox21 in ES cells causes an increase in expression of mesodermal and ectodermal markers followed by a reduced expression of self-renewal genes such as Sox2. The cells continued to express Sox2 but at a lower level,[16] as was the case in our study.

Induced expression of Sox21 resulted in a significant increase of the astrocytic marker S100β in both the subcutaneous and the orthotopical injected tumors. An extensive enhanced expression of CNPase and Tuj1, which are markers for the oligodendrocytic and neuronal lineages, respectively was noticed in the orthotopical injected tumors compared with only a slightly induced expression of CNPase in the subcutaneous tumors. This slight difference between the two in vivo experiments may be due to different environmental conditions, the orthotopical injected tumors are developed in an environment that mimic the native environment of the original tumor as far as possible with neighboring cell types, oxygen and nutrient supply compared with the subcutaneous tumors.[43]

The increased expression of S100β was retained in cell cultures established from the tetracycline-treated tumors after cessation of the treatment, indicating that the Sox21 effect on differentiation is irreversible, at least on a short term basis.

The expression of GFAP, GFAPδ and Nestin in vivo was unchanged irrespective of an induced expression of Sox21. In contrast, in cell cultures, GFAP expression was reduced after Sox21 induction. This finding is consistent with our earlier studies where GFAP expression was downregulated after reduction of Sox2 expression by siRNA in cell lines.[9] The majority of Sox2 positive cells in glioblastoma exhibit coexpression of different lineage markers such as GFAP, Nestin and Tuj1.[34] Whether the concomitant expression of Sox21 is involved in the aberrant differentiation of glioma cells remains to be elucidated but is an interesting possibility. The discrepancy in the in vitro and in vivo results regarding the effect of Sox21 on GFAP expression and the variance in induction of the differentiation marker CNPase and Tuj1 may be related to downstream factors, for example, the availability of the binding site for the Sox protein on DNA[43] and the availability of Sox binding partners[44, 45] or variation in expression levels of Sox2 and Sox21, respectively, in the different experimental set-ups.

Consistent with our previous results there is a significant increase of cleaved caspase-3 in the subcutaneous injected tumors, which suggests an increase in apoptotic cells in the tumors with induced expression of Sox21. There was also a tendency of lower proliferation in the tetracycline/doxycycline-treated mice.

Taken together our results show that induced expression of Sox21 in glioma cells causes reduced tumor size and extended survival. This appears to be a combined effect of induced abnormal differentiation of the tumor cells, induced apoptosis and reduced cell proliferation. Induced expression of Sox21 results in an increased complex binding of Sox2 in vivo, suggesting that these effects are the result of a shift in the balance of available/active Sox2 and Sox21, which most likely results in different expression patterns of targeted genes. The finding that the tumor growth is significantly decreased upon Sox21 induction suggests that Sox21 can act as a tumor suppressor and further this indicates that the Sox2:Sox21 axis may be a potential target for novel therapeutic treatments of Sox2-dependent gliomas.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The authors thank the former students Dakai Yang, Maria Jamalpour, Afsoon Azadi, Elin Sjöberg and Riina Kaukonen for skillful technical assistance. They thank Lene Uhrbom and Karin Forsberg-Nilsson at the Department of Immunology, Genetics and Pathology at Uppsala University for providing the glioblastoma stem cell lines; U-3005/3013/3024/3031/3047 MG.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
ijc28147-sup-0001-suppinfoFig01.tif13082KSupporting Information Fig. 1
ijc28147-sup-0002-suppinfoFig02.tif20825KSupporting Information Fig. 2
ijc28147-sup-0003-suppinfoFig03.tif19877KSupporting Information Fig. 3
ijc28147-sup-0004-suppinfoFig04.tif12602KSupporting Information Fig. 4
ijc28147-sup-0005-suppinfoFig05.tif12958KSupporting Information Fig. 5
ijc28147-sup-0006-suppinfoFig06.tif15031KSupporting Information Fig. 6
ijc28147-sup-0007-suppinfoFig07.tif17933KSupporting Information Fig. 7

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