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. 2013 Nov;19(11):1518-23.
doi: 10.1038/nm.3328. Epub 2013 Sep 29.

Coordinate activation of Shh and PI3K signaling in PTEN-deficient glioblastoma: new therapeutic opportunities

Mariella Gruber Filbin  1 , Sukriti K Dabral, Maria F Pazyra-Murphy, Shakti Ramkissoon, Andrew L Kung, Ekaterina Pak, Jarom Chung, Matthew A Theisen, Yanping Sun, Yoko Franchetti, Yu Sun, David S Shulman, Navid Redjal, Barbara Tabak, Rameen Beroukhim, Qi Wang, Jean Zhao, Marion Dorsch, Silvia Buonamici, Keith L Ligon, Joseph F Kelleher, Rosalind A Segal
Affiliations

Coordinate activation of Shh and PI3K signaling in PTEN-deficient glioblastoma: new therapeutic opportunities

Mariella Gruber Filbin et al. Nat Med. 2013 Nov.

Abstract

In glioblastoma, phosphatidylinositol 3-kinase (PI3K) signaling is frequently activated by loss of the tumor suppressor phosphatase and tensin homolog (PTEN). However, it is not known whether inhibiting PI3K represents a selective and effective approach for treatment. We interrogated large databases and found that sonic hedgehog (SHH) signaling is activated in PTEN-deficient glioblastoma. We demonstrate that the SHH and PI3K pathways synergize to promote tumor growth and viability in human PTEN-deficient glioblastomas. A combination of PI3K and SHH signaling inhibitors not only suppressed the activation of both pathways but also abrogated S6 kinase (S6K) signaling. Accordingly, targeting both pathways simultaneously resulted in mitotic catastrophe and tumor apoptosis and markedly reduced the growth of PTEN-deficient glioblastomas in vitro and in vivo. The drugs tested here appear to be safe in humans; therefore, this combination may provide a new targeted treatment for glioblastoma.

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Figures

Figure 1
Figure 1. Coordinate activation of Shh and PI3k pathways in PTEN-deficient Glioblastoma
(a) gli1, gli2 expression in pten-deficient and –expressing GBMs; robust z-scores for gli1, gli2 (LBL database, 40% cutoff). (*) p = 0.015, (**) p = 1.5 x 10−6. (b) gli2 expression and pten copy number (Broad database ). (*) p = 0.0002 (Supplementary Figure 1b). (c) PTEN Immunoblot in GBMs. Actin: loading control. (d) hBT70, hBT75 GBM neurospheres after treatment with Vehicle, LDE225 (1 μM), BKM120 (100 nM), or combination. Scale bar = 1 mm. (e) Quantification of hBT70 viability in (d). n = 3; two-way ANOVA factorial interaction to identify synergy in e-h, j. F = 5.19, DFn = 1, DFd = 8, p = 0.0523. (*) F = 5.98, DFn = 1, DFd = 8, p = 0.0403. (f) Viability of PTEN-deficient lines (hBT70, 112, 145) and therapies, n = 3; (*) F = 10.27, DFn = 1, DFd = 8, p = 0.0125. (g) Viability of PTEN-expressing lines (hBT75, 188, 239) and therapies. n = 3; F = 0.09, DFn = 1, DFd = 8, p = 0.7749 (n.s. = not significant). (h) PTEN Immunoblot in GBM cells. Actin: loading control. (i) pten, gli1, gli2 mRNA normalized to gapdh. Results normalized to no virus, * = p < .05, ** = p < .01, *** = p < .005, † = 0.06. hBT188 cells (n = 8), For sh-control: p = 0.36, 0.72, 0.20 for pten, gli1, gli2, respectively; for sh-PTEN1 p = 0.03, 1.1x10−12, 0.0004 for pten, gli1, gli2; for sh-PTEN2 p = 0.03, 0.0003, 9x10−6 for pten, gli1, gli2,. hBT239 cells (n = 6), For sh-control p = 1.3, 1.1, 2.72 for pten, gli1, gli2, respectively; sh-PTEN1 p = 0.03, 0.0002, 0.008 for pten, gli1, gli2,; for sh-PTEN2 p = 0.03, 4.3x10−14, 0.06 for pten, gli1, gli2,. Z-test with Bonferroni post-test, comparison to one. (j) GBM response to therapies after pten knock-down hBT188 cells, n = 10, two technical replicates each in five experiments (sh-PTEN1: (*) F = 4.69, DFn = 6, DFd = 81, p = 0.004; sh-PTEN2: (*) F = 3.10, DFn = 6, DFd = 81, p = 0.008): hBT239 cells, n = 9, two technical replicates in three experiments (sh-PTEN1: (*) F = 2.59, DFn = 6, DFd = 72, p = 0.02; sh-PTEN2: (*) F = 2.47, DFn = 6, DFd = 72, p = 0.03). Error bars; ± S.E.M.
Figure 2
Figure 2. Combination of PI3K- and Smo-inhibition reduces tumor growth in vivo
(a) Relative bioluminescence during 80 d of treating intracranial luciferase-expressing hBT112 (n = 10 mice/group). Daily vehicle, LDE225 (60 mg kg−1), BKM120 (30 mg kg−1), or combination therapy. (b) Relative bioluminescence during 100 d of treatment in mice with luciferase-expressing hBT145 (n = 10 mice/group). Drugs above, given in cycles of 3 weeks on, 1 week off. Non-takers, non-tumor deaths censored (<1 animal per group). Treatment groups differ by two-way ANOVA (p < 0.0001). Combination treatment differs from BKM120 after Bonferroni post-test (p < 0.0001). (c) Kaplan-Meier analysis shows increased survival with combination therapy or BKM120, (p < 0.0001) by two-way ANOVA with Bonferroni post-test. (d) Relative bioluminescence of combination therapy and BKM120 treated mice on day 145 (BKM120, combination groups: 7 mice each), day 162 (BKM120: 4 mice; combination: 6 mice). (*) p = 0.03, (**) p = 0.01 by t-tests. (e) MRIs on day 65 (vehicle, LDE225), day 77 (BKM120, combination). 3-D reconstructions; tumor tissue (blue), arrowhead: anterior, tumor volumes indicated. (f) Immunostaining for human NuMA shows tumor cells within mouse brain (day 65 for vehicle, LDE225, day 80 for BKM120, combination). Scale bar = 5mm.
Figure 3
Figure 3. Cellular basis for efficacy of combination treatment
(a) Time-lapse imaging (120 h) of hBT70 cells. Time after drug application in hh:mm:ss, Scale bar = 25μm. (b) Data from 60 cells per condition, three technical replicates, p = 0.0045 by one-way ANOVA. (*) p < 0.05 with Bonferroni post-test. (c) Immunostaining for activated caspase-3 in hBT70, DAPI nuclear staining, Scale bar = 50μm. (d) Apoptotic index increased in combination therapy, n = 3; p < 0.0001 by one-way ANOVA, (*) p < 0.05 with Bonferroni post-test. (e) TUNEL staining in hBT112 xenografts, DAPI nuclear staining, Scale bar = 50μM. (f) Apoptotic index in vivo increased in combination therapy, N ≥ 2000 cells per condition, p < 0.0001 by one-way ANOVA, (*) p < 0.05 after Bonferroni post-test. (g) hBT70 treated, stained for DAPI (blue), γ tubulin (centrosomes; green), acetylated tubulin (spindles; red). Monopolar spindle (right) and normal, bipolar spindle (left). Scale bar = 10μm. (h) Monopolar spindles increased with combination therapy. ≥ 261 cells per treatment, in two independent experiments, (*) p < 0.001 by chi-square. (i) Immunostaining for human NuMA in hBT112 xenografts. Arrowheads indicate abnormal mitoses, Scale bar = 50μM. Error bars; ± S.E.M.
Figure 4
Figure 4. Combination therapy targets PI3k, Shh and S6k pathways
(a) Immunoblots of phosphorylated and total AKT in hBT112 treated with LDE225 (1 μM), BKM120 (500 nM), combination therapy, Actin: loading control. (b) gli1, ptch1 mRNA levels normalized to gapdh (n = 3): Error bars; +/− SEM, p < 0.05 by one-way ANOVA, Tukey post-test. (c) gli1 mRNA levels normalized to gapdh (n = 3): Error bars; +/− SEM, (*) p ≤ 0.005 by z-test compared to one: (**) p < 0.05 by one-way ANOVA, Tukey post-test. (d) Combination treatment with RAD001 (1 nM, 10 nM, 50 nM), and LDE225 (1 μM) synergistically reduces viability of hBT70 (n = 3), two-way ANOVA factorial interaction (*) F = 10.45, DFn = 3, DFd = 16, p = 0.0005. (e) Immunoblots of phosphorylated and total S6K, S6 from hBT112, Actin: loading control. (f) Immunostaining of phosphorylated S6 (pS6) in hBT112 xenografts after 65 d (vehicle, LDE225), 77 days treatment (BKM120, combination). Scale bar = 50μm. (g) pS6-positive cells decreases with LDE225, combination therapy in vivo. Data represent ≥ 900 cells per condition, (*) p = 0.0003 by chi-square, Bonferroni post-test. (h) SAG increases pS6 intensity per cell increases in hBT112. Means of ≥ 1944 cells from three independent experiments. (*) p = 0.01 by one-way ANOVA with Tukey post-test. (i) hBT112 shows decreased viability (n = 3): (*) p < 0.05 by one-way ANOVA with Bonferroni post-test. (j) Model of interaction between PI3K, Shh pathways in PTEN-deficient GBMs. Error bars; ± S.E.M.
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