Male predominance in GBM is subtype specific. Based on their gene expression profiles, GBMs can be divided into classical, mesenchymal, neural, and proneural subtypes (8). The Cancer Genome Atlas (TCGA) data set, as originally reported by Verhaak et al. (8), indicates that mesenchymal and neural subtypes of GBM occur in at least twice as many men as women (Table 1). To determine whether similar sex differences are evident in other large GBM data sets, we developed methods for sex assignment based on the genetic signature of the sex chromosomes that could be applied to publically accessible gene expression profiling data, which frequently have incomplete or no sex information on the samples (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI71048DS1). We examined 2 additional data sets (GSE16011, ref. 9, and GSE13041, ref. 10) containing 426 GBM specimens. Molecular subtypes were assigned using the k-nearest neighbor method (with consideration of varying k) based on the prototype data (8), and missing sex was inferred based on the genetic signature of sex chromosomes. We found that mesenchymal GBM (Mes-GBM) exhibited significant sex disparity in both data sets, with the overall rate of affected men greater than twice that of women. Also evident was a trend toward significantly different rates in both the neural and proneural subsets, but this was not evident in the classical GBM. These data suggest that the effect of sex in GBM is dependent on molecular subtype.

Table 1 Sex-specific rates of GBM molecular subtypes

Sex-specific models of gliomagenesis. Mes-GBM frequently exhibits loss of neurofibromin (NF1) and TP53 function (8). Moreover, in mouse models of disease, combined loss of Nf1 and p53 is sufficient for gliomagenesis (11), but with a male predominance (12). Thus, in both mice and humans, sex interacts with loss of NF1 and TP53 as a determinant of glioma, but the molecular basis for this effect of sex remains unexplained. To investigate whether the effect of sex on gliomagenesis is independent of the acute actions of sex hormones, we established male and female astrocyte cultures and examined their behavior upon stepwise loss of Nf1 and p53.

Male and female astrocytes were isolated from the neocortices of postnatal day 1 Nf1fl/fl GFAP-Cre mice (13, 14). The sex of resultant cultures was confirmed by expression of the X- and Y-encoded paralogs Jarid1c and Jarid1d (refs. 15, 16, and Figure 1A), and complete loss of NF1 expression was verified by Western blot (Figure 1B). Equal purity of male and female astrocyte cultures was established by their near uniform expression of astrocyte markers (GFAP and aldolase C) and the absence of neuronal (NF200) or oligodendrocyte (CNPase) marker expression (Figure 1C). Male and female Nf1–/– astrocytes were infected with retrovirus encoding a flag-tagged dominant-negative form of p53 (DNp53) and EGFP (DNp53-IRES-EGFP) (17). DNp53-expressing cells were purified by FACS based on EGFP expression (Figure 1D). Western blot analysis indicated that endogenous p53 and the flag-tagged DNp53 construct were similarly expressed in male and female astrocytes (Figure 1E). Equivalent loss of p53 function in male and female Nf1–/– DNp53 astrocytes was validated by the parallel reduction in expression of multiple transcriptional targets of p53, including Bai1, p21 (also known as Waf1 and Cip1), and Gadd45a (Figure 1F).

Figure 1 Preparation of male and female Nf1–/– DNp53 astrocytes. (A) Sex determination of isolated mouse astrocytes by PCR for X- and Y-encoded paralogs Jarid1c and Jarid1d. Shown are results with genomic DNA isolated from adult mouse brain and from 3 independent litters of postnatal day 1 pups. (B) Western blot analysis of NF1 expression in male and female Nf1fl/fl, Nf1–/–, and Nf1–/– DNp53 astrocytes. Actin served as loading control. (C) Purity of astrocyte cultures was assessed by immunofluorescence detection of the astrocyte markers aldolase C (red) and GFAP (green) and the absence of neuronal (NF200) and oligodendrocyte (CNPase) marker expression. Nuclei were counterstained blue with DAPI. (D) Direct fluorescence microscopy of FACS-sorted cells indicated 100% EGFP expression in male and female Nf1–/– DNp53 astrocytes. (E) Western blot analysis indicated equal expression of endogenous p53 and the flag-tagged DNp53 construct (FLAG). Actin served as loading control. (F) PCR for p53 transcriptional targets Bai1, p21, and Gadd45a indicates equal loss of expression in Nf1–/– DNp53 astrocytes. ND, not detected; NS, not significant; M, male; F, female. Scale bar: 100 microns.

After establishing the astrocyte cultures, we first measured the effects of NF1 and p53 loss on growth in vitro. While male and female Nf1–/– astrocytes exhibited similar growth rates, the additional loss of p53 function led to a significantly greater increase in the growth of male astrocytes compared with female astrocytes (Figure 2A). These data indicate that sex may influence the functional consequences of tumor suppressor loss in a cell-intrinsic fashion.

Figure 2 Male predominant in vitro transformation of mouse astrocytes with inactivation of NF1 and p53. (A) In vitro growth of male and female Nf1–/– astrocytes and Nf1–/– DNp53 astrocytes over 4 days. Shown are best fits of an exponential growth curve to all data points (***P = 0.0004, ANOVA). (B) EGF treatment (50 ng/ml) supported colony formation (asterisks) in soft agar with male but not female Nf1–/– DNp53 astrocytes. Scale bar: 200 μm.

Combined in vivo loss of Nf1 and p53 was shown to be sufficient for gliomagenesis in mice (11). To determine whether male and female Nf1–/– DNp53 astrocytes were transformed, we evaluated their anchorage-independent growth in soft agar colony formation assays and their tumorigenic potential in intracranial xenograft assays. Neither male nor female Nf1–/– DNp53 astrocytes formed colonies in soft agar (Figure 2B). Tissue analysis 3 months after intracranial implantation revealed that 2 of 7 mice with male Nf1–/– DNp53 astrocyte implants and 2 of 7 mice with female Nf1–/– DNp53 astrocyte implants had tumors (data not shown). Thus, in this model, sequential loss of NF1 and p53 function is only weakly transforming.

Next, we treated male and female Nf1–/– DNp53 astrocytes with PDGF or EGF. The PDGF and EGF pathways are frequently activated in GBM (8, 18–20). The PDGF receptor was equally expressed in male and female Nf1–/– DNp53 astrocytes, and treatment with PDGF was without sex-specific effects on ERK1/2 and AKT phosphorylation, soft agar colony formation, or intracranial tumorigenesis (Supplemental Figure 2).

The EGFR is amplified or mutationally activated in more than 50% of all GBM (21), and EGF can promote transformation in vitro (22). While EGFR mutational activation and amplification is most common in classical GBM, it also occurs in nearly 10% of Mes-GBM (8). Strikingly, EGF treatment resulted in the frequent formation of large (>100-μm diameter) colonies but only in male soft agar cultures (Figure 2B). The persistence of green fluorescence in male and female cells throughout the experiment indicated that the failure to transform female cells was not due to the loss of DNp53 expression. To determine whether the difference in transformation might be due to differences in EGFR expression or activation, we treated male and female Nf1–/– DNp53 astrocytes with EGF and measured EGF-induced activation of ERK1/2 and AKT by immunohistochemistry and Western blot. Male and female Nf1–/– DNp53 astrocytes possessed comparable levels of and similar cell-to-cell uniformity in EGFR expression and similar patterns of EGF-induced ERK and AKT phosphorylation (Supplemental Figure 3). Together, these data indicate that both male and female Nf1–/– DNp53 astrocytes are transformable but that they can possess different thresholds for in vitro transformation, particularly in response to growth factor stimulation.

To determine whether in vitro sexual dimorphism in transformation would translate to differences in in vivo tumorigenesis, we implanted male and female EGF-treated Nf1–/– DNp53 astrocytes into the brains of both male and female nude mice. In contrast to the minimal tumorigenic potential of untreated and PDGF-treated male and female Nf1–/– DNp53 astrocytes, 100% of recipient mice (14 of 14 male, 8 of 8 female) implanted with male EGF-treated Nf1–/– DNp53 cells developed tumors and succumbed to disease (Figure 3A). Strikingly, only 36% of recipient mice (5 of 14 male and 3 of 8 female) implanted with female EGF-treated Nf1–/– DNp53 cells developed tumors and died (P < 0.0001, log-rank test). Tumors were recognizable in situ by their EGFP fluorescence (Figure 3B), confirming persistent transgene (DNp53) expression. Pathological evaluation diagnosed the tumors as high-grade gliomas based on GFAP expression, nuclear atypia, mitoses, and necrosis (ref. 23 and Figure 3C). Notably, there were no histological distinctions between tumors derived from male and female cells (Supplemental Figure 4, A and B). The presence of viable EGFP-positive cells at the injection sites in brains of asymptomatic mice harboring female Nf1–/– DNp53 implants indicated that graft failure and loss of DNp53 expression were not reasons for the lack of tumor formation (Supplemental Figure 4, C and D).

Figure 3 Male predominant in vivo tumorigenesis occurs irrespective of recipient mouse sex. (A) Intracranial implantation of EGF-treated male (n = 22) and female (n = 22) Nf1–/– DNp53 astrocytes from 3 independent litters resulted in death of 100% of mice receiving male cell implants and 36% of mice receiving female cell implants (***P < 0.0001, log-rank test). (B) Intracranial tumors were recognizable in situ by their EGFP expression (asterisk). (C) Intracranial tumors exhibited features of GBM, including nuclear pleomorphism and pseudopalisading necrosis (asterisk, top row), GFAP positivity, and abundant mitoses (asterisk, bottom row). Scale bar: 20 microns. (D) Male (black arrows) and female (white arrows) EGF-treated Nf1–/– DNp53 astrocytes were implanted into the flanks of male and female mice. Male cells gave rise to more tumors regardless of the recipient mouse sex. (E) Flank tumor volumes measured by calipers at 6 weeks. Each symbol represents an individual tumor. Mean volumes were significantly different (**P = 0.001, Wilcoxon rank test).

The in vivo results suggested that intracranial tumorigenesis was determined by the sex of the implanted cells and not by the sex of the recipient mouse. To directly test whether the sex of the microenvironment and circulating sex hormones influence tumorigenesis, we simultaneously implanted male and female EGF-treated Nf1–/– DNp53 astrocytes into the flanks of 8 male and 9 female NCR nude mice. Twelve of seventeen male implants (71%) formed tumors (Figure 3D) equally in male (5 of 8) and female (7 of 9) recipient mice (P = 0.62, Fisher’s exact test). Six of seventeen female implants (35%) formed tumors, again without difference between male (3 of 8) and female (3 of 9) recipient mice (P = 1, Fisher’s exact test). Moreover, male cells exhibited significantly greater maximal tumor growth (Figure 3E). Thus, in both the intracranial and flank locations, male EGF-treated Nf1–/– DNp53 astrocytes more readily formed tumors than their female counterparts, regardless of the sex of the surrounding tumor microenvironment. These data demonstrate that, in this model, sex-specific thresholds exist for transformation and that cell-intrinsic differences, rather than the sex of the microenvironment, are the stronger determinants of tumor growth.

Mechanisms of sexually dimorphic transformation. To understand why male astrocytes may be more susceptible to malignant transformation, we first measured the clonogenic subpopulation. Loss of p53 function can enhance self-renewal and the reacquisition of a multipotent stem-like cell state (24), which may be essential for malignant transformation of differentiated cells (25). Clonogenic cell frequency, as measured by extreme limiting dilution assays (ELDA) (26), ranged from 6.1% to 17.6% in male Nf1–/– DNp53 astrocytes and 1.6% to 3.4% in female Nf1–/– DNp53 astrocytes (Figure 4, A and B). The greater stem-like cell frequency in male Nf1–/– DNp53 cells was associated with higher expression of stem cell markers CD133 and Sox2 (Figure 4C) as well as the capability of forming larger and greater numbers of neurospheres (Figure 4D).

Figure 4 Sex differences in induction of a stem-like cell subpopulation in Nf1–/– DNp53 astrocytes. (A) Frequency of clonogenic (stem-like) cells was measured by ELDA in Nf1–/– DNp53 cells (3,000, 600, 120, 24, 5, and 1 cell per well; 10–12 replicates per dilution), with 3 independent astrocyte preparations. Shown is a representative ELDA analysis from one of the three cell preparations. CSC, clonogenic (stem-like) cell. (B) Male Nf1–/– DNp53 cells have a significantly higher stem-like cell frequency (12.2% ± 3.4%) than female (2.77% ± 0.6%) counterparts, as derived from the ELDA analysis (*P = 0.03, t test). (C) Sex differences in expression of stem cell markers CD133 and Sox2 (*P = 0.018 and **P = 0.03 for CD133 and Sox2 respectively, 2-tailed t test). (D) Representative images of neurospheres from male and female Nf1–/– DNp53 cells (3,000 cells per well) 1 week after plating. Scale bar: 500 microns. (E) The frequency of clonogenic stem-like cells, as determined by ELDA, was equal in male (2.49% ± 1.1%) and female (1.73% ± 0.63) Nf1–/– NSCs. (F) The frequency of clonogenic stem-like cells, as determined by ELDA, was equally low (0.2%) in both male and female Nf1–/– astrocytes.

Next, we asked whether sex differences in stem-like cell activity were a result of p53 inactivation or whether they preexisted as a consequence of Nf1 loss. The frequency of clonogenic cells in Nf1–/– postnatal day 1 hippocampal neural stem cells (NSCs) was approximately 2%, and no differences were observed between male and female cultures (Figure 4E). To determine whether sex differences might emerge with differentiation, we measured clonogenicity in Nf1–/– astrocytes. Both male and female Nf1–/– astrocytes possessed an equivalently low frequency (0.2%) of cells with clonogenic potential (Figure 4F). Thus, sex differences in clonogenic stem-like cell properties between male and female Nf1–/– DNp53 astrocyte cultures are a result of sexually dimorphic response to the loss of p53 function.

To further dissect the mechanistic basis for sex differences in transformation, we examined rates of apoptosis and proliferation in cultured male and female Nf1–/– DNp53 astrocytes. Apoptosis was measured by quantifying levels of cleaved caspase-3 under basal conditions and after etoposide treatment. While there was a trend toward greater basal and etoposide-induced apoptosis in male Nf1–/– DNp53 astrocytes, this did not reach statistical significance (Figure 5A). To measure proliferation, we quantified the percentage of nuclei positive for phospho-histone H3 (pHH3), a widely used proliferation marker in gliomas (27). We found that male Nf1–/– DNp53 astrocytes exhibited significantly higher levels of pHH3 positivity compared with female Nf1–/– DNp53 astrocytes (Figure 5B), indicating that they possess a higher rate of proliferation.

Figure 5 Sex differences in proliferation of Nf1–/– DNp53 astrocytes. (A) Protein lysates of male and female Nf1–/– DNp53 astrocytes treated with DMSO or etoposide (10 μg/ml, 24 hours) were analyzed by Western blot for cleaved caspase-3 and total caspase-3. Actin served as loading control. A single representative blot and quantitation from the 3 independent experiments are shown. (B) Representative fields from cultures of male and female Nf1–/– DNp53 astrocytes stained with hematoxylin and for the presence of the nuclear proliferation marker pHH3 (brown). Blue arrowheads identify examples of nuclei negative for pHH3. Red arrowheads identify examples of nuclei positive for pHH3. Scale bar: 100 microns. Quantification of the percentage of positive nuclei is shown (*P < 0.05, t test). (C) Representative histograms from male and female Nf1–/– DNp53 astrocyte cell cycle analysis of asynchronously growing serum-supplemented cultures. Quantified areas for each phase of the cell cycle are as indicated. Means for cell cycle distribution from 3 independent cultures are shown.

Next, we performed cell cycle analysis. Under basal asynchronous conditions, the fraction of male Nf1–/– DNp53 astrocytes in the S and G 2 /M phases of the cell cycle was greater than in their female counterparts (Figure 5C). To understand why male Nf1–/– DNp53 astrocytes exhibited greater proliferation and S/G 2 /M cell cycle distribution, we examined regulation of the retinoblastoma protein (RB) pathway. The RB pathway is a negative regulator of proliferation, and RB loss or inactivation is among the most common features of human cancers (28). To assess RB regulation, we serum starved male and female Nf1–/– DNp53 astrocytes to promote arrest in the G 1 phase of the cell cycle and then followed RB phosphorylation (inactivation) and cell cycle progression after adding serum back. Western blot analysis indicated that, under conditions of serum starvation, male and female Nf1–/– DNp53 astrocytes expressed comparable levels of RB and phosphorylated RB (Figure 6, A and B). Upon addition of serum, male Nf1–/– DNp53 astrocytes exhibited significantly greater time-dependent phosphorylation of RB compared with that of female astrocytes.

Figure 6 Sex differences in RB inactivation in Nf1–/– DNp53 astrocytes. (A) Western blot analysis for RB and phospho-RB (p-RB) in protein lysates from cultures of male and female Nf1–/– DNp53 astrocytes serum starved for 48 hours (t = 0) and after addition of serum for the indicated times. Actin served as loading control. Shown are representative blots from 1 of 3 independent experiments. (B) Quantification of Western blot analysis of RB phosphorylation. Shown is the ratio of p-RB/RB as a function of time in serum (***P = 0.0001, ANOVA). (C) RB inactivation was mesured with an E2F-Luc reporter in 4 independent cultures of male and female Nf1–/– DNp53 astrocytes. For each measurement, bioluminescence was normalized to EGFP fluorescence, which was linearly related to cell number in both male and female astrocytes (inset). Male values were normalized to female values within each experiment (*P < 0.05, t test). (D) G 1 fraction obtained from cell cycle analysis of male and female Nf1–/– DNp53 astrocytes cultured in serum or serum starved for 48 hours. Quantitation of 4 independent experiments is shown (*P = 0.028, t test).

RB regulates transition from the G 1 to the S phase of the cell cycle through sequestration of E2F transcription factors. Hyperphosphorylation of RB or loss of RB expression results in increased E2F-dependent transcription and more rapid transit from G 1 to S phase. To determine whether the differences in RB phosphorylation had functional consequences, we examined E2F-dependent transcription and cell cycle distribution in male and female Nf1–/– DNp53 astrocytes upon addition of sera. To measure E2F-dependent transcription, we constructed a lentiviral reporter system with 3XE2F-driven luciferase expression (Supplemental Figure 5) and established stable lines of male and female Nf1–/– DNp53 E2F-Luc reporter cells. Male and female Nf1–/– DNp53 E2F-Luc reporter cells were serum starved for 24 hours and then treated with 10% serum-supplemented media for 6 hours. As cell number was linearly related to EGFP fluorescence (Figure 6C, inset), we were able to normalize E2F-Luc reporter activity to EGFP measures. Consistent with the measured differences in RB phosphorylation, male Nf1–/– DNp53 astrocytes exhibited a greater level of E2F-Luc activity in response to serum (Figure 6C). As expected, sex differences in RB phosphorylation and E2F reporter activity were correlated with differences in cell cycle regulation. While serum starvation significantly increased the G 1 fraction in female Nf1–/– DNp53 astrocytes, it produced no significant G 1 change in male Nf1–/– DNp53 astrocytes, indicating that male cells possessed lower RB activity (Figure 6D). Together, these findings strongly indicate that RB regulation is sexually dimorphic in Nf1–/– DNp53 astrocytes and that the functional consequences of this include greater proliferation in male cells.

To test whether differential inactivation of RB underlies sex differences in transformation, we expressed SV40 large T antigen (SV40-TAg) in male and female Nf1–/– astrocytes (29). SV40-TAg completely inactivates both p53 and RB pathways (reviewed in ref. 30). Quantitative PCR verified equal SV40-TAg expression and equivalent reductions in expression of p53 transcriptional targets p21 and Gadd45a and expected increases and decreases in expression of E2F targets Cdc6 (31) and Bcl2 (32), respectively (Figure 7A). In soft agar assays, we found that, in contrast to EGF-treated Nf1–/– DNp53 astrocytes, both male and female Nf1–/– SV40-TAg astrocytes were competent to form colonies (Figure 7B). Moreover, both male and female Nf1–/– SV40-TAg astrocytes formed tumors of similar sizes upon flank implantation (Figure 7C), regardless of the sex of recipient mice. These data indicate that under conditions of equivalent combined loss of p53 and RB function, male and female astrocytes are equally transformed.