Protein Phosphatase 2A Mediates Dormancy of Glioblastoma Multiforme-Derived Tumor Stem-Like Cells during Hypoxia

The hypoxic microenvironment of glioblastoma multiforme (GBM) is thought to increase resistance to cancer therapies. Recent evidence suggests that hypoxia induces protein phosphatase 2A (PP2A), a regulator of cell cycle and cell death. The effects of PP2A on GBM tumor cell proliferation and survival during hypoxic conditions have not been studied.Expression of PP2A subunits and HIF-α proteins was measured in 65 high-grade astrocytoma and 18 non-neoplastic surgical brain specimens by western blotting. PP2A activity was measured by an immunoprecipitation assay. For in vitro experiments, GBM-derived tumor stem cell-like cells (TSCs) were exposed to severe hypoxia produced by either CoCl₂ or 1% O₂. PP2A activity was inhibited either by okadaic acid or by shRNA depletion of the PP2A C subunit. Effects of PP2A activity on cell cycle progression and cell survival during hypoxic conditions were assessed using flow cytometry.In our patient cohort, PP2A activity was positively correlated with HIF-1∝ protein expression (P = 0.002). Patients with PP2A activity levels above 160 pMP had significantly worse survival compared to patients with levels below this threshold (P = 0.002). PP2A activity was an independent predictor of survival on multivariable analysis (P = 0.009). In our in vitro experiments, we confirmed that severe hypoxia induces PP2A activity in TSCs 6 hours after onset of exposure. PP2A activity mediated G1/S phase growth inhibition and reduced cellular ATP consumption in hypoxic TSCs. Conversely, inhibition of PP2A activity led to increased cell proliferation, exhaustion of intracellular ATP, and accelerated P53-independent cell death of hypoxic TSCs.Our results suggest that PP2A activity predicts poor survival in GBM. PP2A appears to reduce the metabolic demand of hypoxic TSCs and enhances tumor cell survival. Modulation of PP2A may be a potential target for cancer therapy

Glioblastoma Stem-Like Cells—Biology and Therapeutic Implications

The cancer stem-cell hypothesis proposes that malignant tumors are likely to encompass a cellular hierarchy that parallels normal tissue and may be responsible for the maintenance and recurrence of glioblastoma multiforme (GBM) in patients. The purpose of this manuscript is to review methods for optimizing the derivation and culturing of stem-like cells also known as tumor stem cells (TSCs) from patient-derived GBM tissue samples. The hallmarks of TSCs are that they must be able to self-renew and retain tumorigenicity. The isolation, optimization and derivation of TSCs as outlined in this review, will be important in understanding biology and therapeutic applications related to these cells

Tight regulation between cell survival and programmed cell death in GBM stem-like cells by EGFR/GSK3b/PP2A signaling

Malignant gliomas represent one of the most aggressive forms of cancer, displaying high mortality rates and limited treatment options. Specific subpopulations of cells residing in the tumor niche with stem-like characteristics have been postulated to initiate and maintain neoplasticity while resisting conventional therapies. The study presented here aims to define the role of glycogen synthase kinase 3 beta (GSK3b) in patient-derived glioblastoma (GBM) stem-like cell (GSC) proliferation, apoptosis and invasion. To evaluate the potential role of GSK3b in GBM, protein profiles from 68 GBM patients and 20 normal brain samples were analyzed for EGFR-mediated PI3kinase/Akt and GSK3b signaling molecules including protein phosphatase 2A (PP2A). To better understand the function of GSK3b in GBM, GSCs were isolated from GBM patient samples. Blocking GSK3b phosphorylation at Serine 9 attenuated cell proliferation while concomitantly stimulating apoptosis through activation of Caspase-3 in patient-derived GSCs. Increasing GSK3b protein content resulted in the inhibition of cell proliferation, colony formation and stimulated programmed cell death. Depleting GSK3b in GSCs down regulated PP2A. Furthermore, knocking down PP2A or blocking its activity by okadaic acid inactivated GSK3b by increasing GSK3b phosphorylation at Serine 9. Our data suggests that GSK3b may function as a regulator of apoptosis and tumorigenesis in GSCs. Therapeutic approaches targeting GSK3b in glioblastoma stem-like cells may be a useful addition to our current therapeutic armamentarium

PP2A inhibition enhances cell death of hypoxic TSCs.

<p>(A) Examples of Annexin/PI stainings of TSCs grown for seven days in either normal culturing conditions or in media supplemented with 200 µM of CoCl₂. Addition of 1 nM of OA has no significant effect on cells propagated in normal culturing conditions, but significantly increases the number of Annexin V+/PI+ cells in hypoxic conditions. (B) Photographs of TSCs <i>in vitro</i> following one week of culture during normoxia or hypoxia with or without inhibition of PP2A. Viable cells are stained by formazan dye. Few viable cells (black, arrows) are seen in hypoxic cells exposed to OA. (C) While neither 0.5 nM nor 1 nM of OA exerts a significant effect on the death of TSCs in routine culturing conditions, OA significantly increases the amount of Annexin V+/PI+ TSCs during hypoxic conditions. Bars represent the mean value of 6 independent experiments ± SEM. (D) Depletion of the PP2A-C causes an almost two-fold increase of cell death during hypoxic conditions. Importantly, enhancement of PP2A activity by PME-1 depletion reduces death of hypoxic TSCs to levels comparable to normoxia. (E) Inhibition of PP2A in TSCs exposed to hypoxic conditions for 3 days leads to increased phosphorylation of PLK and AKT.</p

PP2A reduces ATP consumption and inhibits growth of TSCs during hypoxic conditions.

<p>(A) Effect of PP2A inhibition on ATP consumption of TSCs during normoxia and hypoxia. Intracellular ATP was measured in TSCs 1 hour following substrate depletion (D5030 media) with our without PP2A inhibition. PP2A inhibition leads to significantly higher ATP consumption during hypoxic conditions. The ATP content was measured and normalized to the lysate protein content. Bars represent the mean values of 8 independent experiments ± SEM. (B) 24 hours following plating, TSCs are exposed to CoCl₂ and/or OA for additional 24 hours. Hypoxia leads to G1/S phase growth inhibition in TSCs. Representative examples of cell cycle analysis experiments. (C) Summary of six independent experiments reveals that hypoxia-mediated G1/S phase arrest is partially reversed by OA in a dose-dependent fashion. Accordingly, PP2A inhibition allows for significantly more hypoxic TSCs to progress into the S-phase. (D) In line with pharmacological inhibition of PP2A, depletion of PP2A by shRNA alleviates G1/S cell cycle arrest in TSCs propagated in CoCl₂. Given the longer duration of PP2A inhibition in PP2A-depleted cells (48 h) compared to OA-treated cells (24 h [C]), more PP2A-depleted cells progress to G2 phase. Increasing PP2A activity by depletion of the endogenous PP2A inhibitor PME-1 has the opposite effect. Western blots reveal depletion of PP2A-C and PME-1 in TSCs by shRNA.</p

GBM Patient Characteristics (n = 60).

<p>GBM Patient Characteristics (n = 60).</p

Hypoxia increases cyclin G2 expression and PP2A activity in TSCs.

<p>(A) At 2, 4, 6, 8, 12, and 24 hours following addition of CoCl₂ to culture media, HIF-1α protein is detected in TSCs while cells grown in standard conditions lack expression. Increased cyclin G2 levels are detected in TSCs six hours following exposure to CoCl₂. Expression levels of the PP2A C subunit remain stable throughout the experiment. Bar graph depicts marked increase of PP2A activity following exposure of TSCs to CoCl₂. Thus, PP2A activity levels are nine times higher in TSCs propagated in CoCl₂ for six hours compared to TSCs grown in standard conditions. Bars represent the mean value of 3 independent experiments ± SEM. (B) To determine whether PP2A directly interacts with cyclin G2, PP2A was immunoprecipitated in cell lysates from control TSCs and TSCs exposed to six hours of CoCl₂. In both conditions, the PP2A C subunit forms a complex with cyclin G2.</p

Cox multivariate analysis for survival prediction.

<p>Cox multivariate analysis for survival prediction.</p