A molecular cascade modulates MAP1B and confers resistance to mTOR inhibition in human glioblastoma

Abstract

Background: Clinical trials of therapies directed against nodes of the signaling axis of phosphatidylinositol-3 kinase/Akt/mammalian target of rapamycin (mTOR) in glioblastoma (GBM) have had disappointing results. Resistance to mTOR inhibitors limits their efficacy.

Methods: To determine mechanisms of resistance to chronic mTOR inhibition, we performed tandem screens on patient-derived GBM cultures.

Results: An unbiased phosphoproteomic screen quantified phosphorylation changes associated with chronic exposure to the mTOR inhibitor rapamycin, and our analysis implicated a role for glycogen synthase kinase (GSK)3B attenuation in mediating resistance that was confirmed by functional studies. A targeted short hairpin RNA screen and further functional studies both in vitro and in vivo demonstrated that microtubule-associated protein (MAP)1B, previously associated predominantly with neurons, is a downstream effector of GSK3B-mediated resistance. Furthermore, we provide evidence that chronic rapamycin induces microtubule stability in a MAP1B-dependent manner in GBM cells. Additional experiments explicate a signaling pathway wherein combinatorial extracellular signal-regulated kinase (ERK)/mTOR targeting abrogates inhibitory phosphorylation of GSK3B, leads to phosphorylation of MAP1B, and confers sensitization.

Conclusions: These data portray a compensatory molecular signaling network that imparts resistance to chronic mTOR inhibition in primary, human GBM cell cultures and points toward new therapeutic strategies.

Fig. 1

Phosphoproteomics of mTOR inhibition. (A) Heatmap of phosphoproteomics results comparing chronic rapamycin treatment or acute rapamycin treatment with DMSO treated controls. The relative levels of phosphopeptides are expressed as log2 of the ratios relative to control (DMSO). The heatmap on the left depicts proteins with ≥2-fold differences and the one on the right shows those with ≥4-fold differences. (B) Western blot of phosphorylated 4EBP1-T37/46 (p4EBP1), phosphorylated RB1-S249 (pRB1), and phosphorylated RPS6-S240/244 (pS6) demonstrating changes after 14 days of chronic rapamycin treatment (14 days, 100 nM). Phosphoproteomics values (−2.28x and +2.17x) above blots indicate the fold changes for p4EBP1-T46 and pRB1-S249, respectively, in phosphorylation levels as detected by the phosphoproteomics screen. See also Supplementary Table S1.

Fig. 2

GSK3B inhibition confers resistance to mTOR pathway inhibition. (A) Dose response to a serial dilution of rapamycin with co-treatment of the GSK3B inhibitor CHIR99021 (1 μM) (P = 0.0054, Mann–Whitney test). (B) Dose response to a serial dilution of BEZ235 with co-treatment of the GSK3B inhibitor CHIR99021 (1 μM), P < 0.0001 comparing IC₅₀ values. (C) Western blot of HK301 cells after 2 hours treatment with DMSO, rapamycin (100 nM), CHIR99021 (4 μM), or rapamycin (100 nM) + CHIR99021 (4 μM). The top band of 3 bands in the phosphorylated 4EBP1, indicated by the arrow, is for threonine-46. (D) Western blot of GSK3B knockdown demonstrates specificity for GSK3B with no depletion of GSK3A. (E) Fitted curve of log-transformed values for a serial dilution of rapamycin in HK301 GBM cells with and without GSK3B knockdown, P < 0.0001 comparing IC₅₀ values. (F) Fitted curve of log transformed values for a serial dilution of BEZ235 in HK301 GBM cells with and without GSK3B knockdown. P = 0.0007 comparing IC₅₀ values. N = 3 independent experiments for A, B, E, and F. See also Supplementary Figures S1 and S2.

Fig. 3

Depletion of mTORC1 and, in certain GBM cultures, mTORC2 can promote inhibitory phosphorylation of GSK3B. (A) Western blot of GBM cultures HK157 and HK301 demonstrating efficacy of knockdown and phosphorylation status of GSK3B, ERK, and Akt. (B) Mean relative cell number and standard error bars for a serial dilution of rapamycin treatments in HK157 in the presence or absence of shRICTOR, P = 0.0004 comparing mean IC₅₀ values (N = 3, log scale). (C) Mean relative cell number and standard error bars for a serial dilution of BEZ235 treatments in HK157 in the presence and absence of shRICTOR, P < 0.001 comparing IC₅₀ values (N = 3, log scale). (D) Mean relative cell number and standard error bars for a serial dilution of rapamycin treatments in HK301 in the presence and absence of shRICTOR (P = 0.8618) is depicted comparing IC₅₀ values (N = 2, log scale). (E) Mean relative cell number and standard error bars for a serial dilution of BEZ235 treatments in HK301 in the presence and absence of shRICTOR, P = 0.1012 comparing IC₅₀ values (N = 2, log scale). See also Supplementary Figure S3.

Fig. 4.

Knockdown of MAP1B confers sensitivity to chronic mTOR inhibition. (A) Fluorescent images of wells from 384 well plates used in the targeted shRNA screen display green fluorescent protein–labeled cells in shMAP1B conditions at 10 days posttreatment (DMSO-control or 100 nM rapamycin [RAPA]). White scale bar represents 2.67 mm. (B) Fitted curve of log transformed values for a serial dilution of rapamycin in HK217 GBM cells, with and without MAP1B knockdown, after 6 days of proliferation, P = 0.0002 comparing IC₅₀ values (N = 3). (C) Fitted curve of log transformed values for a serial dilution of BEZ235 in HK217 GBM cells, with and without MAP1B knockdown, after 6 days of proliferation, P = 0.0028 comparing IC₅₀ values (N = 4). (D) Western blot of HK301 cells. Western blot columns are in order: 7 day chronic DMSO-control, 7 day rapamycin, shRNA-control (C), shMAP1B (M), shGSK3B (G). (E) Observed points, standard error bars, and fitted lines for normalized tumor volumes of HK374 subcutaneous tumors at different time intervals. By day 12 posttreatment, the shMAP1B under rapamycin treatment demonstrates increased sensitivity and reduced tumor growth, as indicated by a comparison of the slopes (P < 0.0001) as well as by comparison of the means (P = 0.0323, Kruskal‒Wallis test). (F) Western blot of the tumor lysates demonstrates that the shMAP1B transplanted tumors maintained depletion of MAP1B throughout the tumor growth. Phosphorylated RPS6 levels demonstrate that rapamycin inhibited its target pathway in the tumors. (G) Bar graph shows mean ± SEM for the corrected total cell fluorescence (CTCF) of alpha-tubulin in HK217 GBM cells after 7 days of chronic rapamycin treatment followed by acute exposure to 5 μM Nocodazole (30 min). There is a significant increase in the rapamycin conditions compared with the DMSO conditions for the pGIPZ-Ctrl vector (P = 0.001; Mann‒Whitney test). There is no significant difference between the DMSO and rapamycin conditions for the shMAP1B infected cells (P = 0.1431). Each condition represents the corrected total cell fluorescence of 10 individual cells. (H) Representative photographs of alpha tubulin staining of HK217 GBM cells in the different 7 day treatment conditions and after acute Nocodazole treatment. White scale bar indicates 10 μM. See also Supplementary Table S2, and Supplementary Figures S4, S5.

Fig. 5

In vivo support for our model of regulation of resistance to mTOR inhibition. (A) Representative sample of sensitization effect on HK374. Log scale of relative cell numbers following treatment with a serial dilution of doses of selumetinib (μM) in the presence of DMSO (black line), 100 nM rapamycin (red line), or 10 nM BEZ235 (green line). The calculated IC₅₀ values are displayed for each combinatorial treatment (selumetinib + second inhibitor listed). (B) Quantification of tumor value at day 9 is depicted. Treatment groups (mean ± SEM) are compared with the DMSO treated control. P-values for the Mann‒Whitney statistical comparison test are indicated for each comparison. ANOVA comparing all groups had P = 0.0010. (C) Western blot of pooled xenograft tumors (6 animals/treatment group, 12 tumors/treatment group) depicts effective targeting of rapamycin and selumetinib after 9 days of treatment as well as phosphorylation status of MAP1B and GSK3B. (D) Schematic of proposed model of resistance to chronic mTOR inhibition. Dotted line represents indirect association. Red color indicates inactivation under chronic treatment, green color indicates activation. See also Supplementary Figure S6.