OSU-03012 suppresses GRP78/BiP expression that causes PERK-dependent increases in tumor cell killing

Abstract

We have further defined mechanism(s) by which the drug OSU-03012 (OSU) kills tumor cells. OSU lethality was suppressed by knock down of PERK and enhanced by knock down of ATF6 and IRE1α. OSU treatment suppressed expression of the chaperone, BiP/GRP78, and did so through reduced stability of the protein. Knock down of BiP/GRP78 further enhanced OSU lethality. Overexpression of BiP/GRP78 abolished OSU toxicity. Pre-treatment of cells with OSU enhanced radiosensitivity to a greater extent than concomitant or sequential drug treatment with radiation exposure. Expression of a mutant active p110 PI3K, or mutant active forms of the EGFR in GBM cells did not differentially suppress OSU killing. In contrast loss of PTEN function reduced OSU lethality, without altering AKT, p70 S6K or mTOR activity, or the drug's ability to radiosensitize GBM cells. Knock down of PTEN protected cells from OSU and radiation treatment whereas re-expression of PTEN facilitated drug lethality and radiosensitization. In a dose-dependent fashion OSU prolonged the survival of mice carrying GBM tumors and interacted with radiotherapy to further prolong survival. Collectively, our data show that reduced BiP/GRP78 levels play a key role in OSU-3012 toxicity in GBM cells, and that this drug has in vivo activity against an invasive primary human GBM isolate.

Figure 1

OSU-03012 toxicity in transformed cells is regulated by ER stress signaling proteins. (A) Upper: GBM12 cells were treated with 2 µM OSU-03012 and the expression of LC3I and LC3II determined over a 24 h time-course. Lower: GBM12 cells were transfected with empty vector plasmid (CMV) or a plasmid to express dominant negative PERK. Twenty-four hours after transfection cells were treated with vehicle (DMSO) or with OSU-03012 (2 µM). Cells were isolated after 6 h and the levels of LC3II determined. (B) GBM6 cells were transfected with scrambled siRNA (siSCR) or siRNAs to knock down ATG5 or Beclin1. Twenty-four hours after transfection cells were treated with vehicle (DMSO) or 2 µM OSU-03012. Viability was determined 48 h later (n = 3, +/- SEM). (C) GBM6 cells were transfected with scrambled siRNA (siSCR) or siRNAs to knock down PERK, ATF6 or IRE1α. Twenty-four hours after transfection cells were treated with vehicle (DMSO) or 2 µM OSU-03012. Cells were then irradiated (4 Gy). Viability was determined 48 h later (n = 3, +/- SEM). (D) GBM6 cells were transfected with empty vector plasmid (CMV) or a plasmid to express dominant negative PERK. Twenty-four hours after transfection cells were treated with vehicle (DMSO) or with OSU-03012 (2 µM). Viability was determined 48 h later (n = 3, +/- SEM).

Figure 2

OSU-03012 radiosensitizes GBM cells. (A) GBM6 cells were treated with vehicle (DMSO) or with OSU-03012 (2 µM) and 30 min later irradiated (2 Gy, 4 Gy). Viability was determined 24 and 48 h later (n = 3, +/- SEM). (B) GBM12 cells were treated with vehicle (DMSO) or with OSU-03012 (2 µM) and 30 min later irradiated (2 Gy, 4 Gy). Viability was determined 24 and 48 h later (n = 3, +/- SEM). (C) GBM6 and GBM12 cells were plated as single cells. Twelve hours after plating cells were treated with vehicle or OSU-03012 (2 µM) and 30 min later irradiated (4 Gy). Forty-eight hours later the media was replaced with drug free media. Colonies were permitted to form over the following ∼14 d. Colonies of > 50 cells were counted (n = 3, +/- SEM). (D) GBM6 and GBM12 cells were treated with vehicle (DMSO) or with 2 µM OSU-03012 for 24 h (pre-treatment: “pre”); after which time portions of the vehicle treated cells were treated with OSU-03012 (concomitant: “concom”). Cells were irradiated (2 Gy, 4 Gy). In parallel sets, cells were incubated with vehicle for 48 h, irradiated and OSU-03012 added 24 h after irradiation. All cells were isolated for viability 24 h after radiation exposure. Data are presented as the true percentage increase above control values for each condition (n = 3, +/- SEM). (E) Left: SW480 and SW620 cells were treated with increasing doses of OSU-03012 (0–3 µM). Cells were isolated 48 h after drug treatment and viability determined (n = 3, +/- SEM); Right: SW480 and SW620 cells were treated with OSU-03012 (2 µM). Cells were irradiated and then isolated 48 h after drug treatment and viability determined (n = 3, +/- SEM). (F) Wild-type SW620 and SW620 cells with restored LASS6 expression were treated with OSU-03012 (2 µM). Cells were irradiated and then isolated 48 h after drug treatment and viability determined (n = 3, +/- SEM).

Figure 2

OSU-03012 radiosensitizes GBM cells. (A) GBM6 cells were treated with vehicle (DMSO) or with OSU-03012 (2 µM) and 30 min later irradiated (2 Gy, 4 Gy). Viability was determined 24 and 48 h later (n = 3, +/- SEM). (B) GBM12 cells were treated with vehicle (DMSO) or with OSU-03012 (2 µM) and 30 min later irradiated (2 Gy, 4 Gy). Viability was determined 24 and 48 h later (n = 3, +/- SEM). (C) GBM6 and GBM12 cells were plated as single cells. Twelve hours after plating cells were treated with vehicle or OSU-03012 (2 µM) and 30 min later irradiated (4 Gy). Forty-eight hours later the media was replaced with drug free media. Colonies were permitted to form over the following ∼14 d. Colonies of > 50 cells were counted (n = 3, +/- SEM). (D) GBM6 and GBM12 cells were treated with vehicle (DMSO) or with 2 µM OSU-03012 for 24 h (pre-treatment: “pre”); after which time portions of the vehicle treated cells were treated with OSU-03012 (concomitant: “concom”). Cells were irradiated (2 Gy, 4 Gy). In parallel sets, cells were incubated with vehicle for 48 h, irradiated and OSU-03012 added 24 h after irradiation. All cells were isolated for viability 24 h after radiation exposure. Data are presented as the true percentage increase above control values for each condition (n = 3, +/- SEM). (E) Left: SW480 and SW620 cells were treated with increasing doses of OSU-03012 (0–3 µM). Cells were isolated 48 h after drug treatment and viability determined (n = 3, +/- SEM); Right: SW480 and SW620 cells were treated with OSU-03012 (2 µM). Cells were irradiated and then isolated 48 h after drug treatment and viability determined (n = 3, +/- SEM). (F) Wild-type SW620 and SW620 cells with restored LASS6 expression were treated with OSU-03012 (2 µM). Cells were irradiated and then isolated 48 h after drug treatment and viability determined (n = 3, +/- SEM).

Figure 3

OSU-03012 is a more effective anticancer drug than Celecoxib. Panel (A) Lower: GBM6 and GBM12 cells were treated with increasing concentrations of Celecoxib (0–20 µM) or with OSU-03012 (2 µM). Cells were isolated 24 and 48 h later and viability determined (n = 3, +/- SEM). Upper: OSU-03012 reduces BiP/GRP78 expression whereas Celecoxib increases BiP/GRP78 levels. Cells were treated with OSU-03012 (1–2 µM) or Celecoxib (5–20 µM) and cells isolated 24 h after drug treatment. (B) GBM12 cells were treated with vehicle (DMSO), OSU-03012 (2 µM) or Celecoxib (10 µM). Cells were irradiated 30 min after drug treatment and portions of cells isolated 24 and 48 h later for viability determination (n = 3, +/- SEM).

Figure 4

Knock down of BiP/GRP78 enhances OSU-03012 toxicity. (A) GBM12 cells were transfected with scrambled siRNA or with an siRNA to knock down BiP/GRP78. Twenty-four hours after transfection cells were treated with vehicle (DMSO) or OSU-03012 (2 µM). Portions of cells were isolated 24 and 48 h later for viability determination (n = 3, +/- SEM). (B) GBM6 cells were transfected with scrambled siRNA or with an siRNA to knock down BiP/GRP78. Twenty-four hours after transfection cells were treated with vehicle (DMSO) or OSU-03012 (2 µM). Portions of cells were isolated 24 h and 48 h later for viability determination (n = 3, +/- SEM). (C) GBM14 cells were transfected with scrambled siRNA or with an siRNA to knock down BiP/GRP78. Twenty-four hours after transfection cells were treated with vehicle (DMSO) or OSU-03012 (2 µM). Portions of cells were isolated 24 and 48 h later for viability determination (n = 3, +/- SEM).

Figure 5

OSU-03012 lethality is suppressed by overexpression of BiP/GRP78. GBM cells, as indicated, were transfected with empty vector plasmid (CMV) or with a plasmid to express BiP/GRP78. Twenty-four hours after transfection cells were treated with vehicle (DMSO) or OSU-03012 (2 µM). Portions of cells were isolated 24 and 48 h later for viability determination (n = 3, +/- SEM). Upper blots in (A and B) show that expression of exogenous plasmid derived BiP/GRP78 is inhibited by OSU-03012.

Figure 6

Loss of BiP/GRP78 does not further radiosensitize GBM cells above the sensitization effect caused by OSU-03012. GBM cells as indicated were transfected with scrambled siRNA or with an siRNA to knock down BiP/GRP78. Twenty-four hours after transfection cells were treated with vehicle (DMSO) or OSU-03012 (2 µM). Cells were irradiated (4 Gy). Cells were isolated 24 h later for viability determination (n = 3, +/- SEM). The arrows with numeric value indicate the percentage increase in death above each corresponding vehicle control.

Figure 7

Overexpression of BiP/GRP78 suppresses OSU-03012-induced radiosensitization. GBM cells, as indicated, were transfected with empty vector plasmid (CMV) or with a plasmid to express BiP/GRP78. Twenty-four hours after transfection cells were treated with vehicle (DMSO) or OSU-03012 (2 µM). Cells were then irradiated. Cells were isolated 24 h later for viability determination (n = 3, +/- SEM). The arrows with numeric value indicate the percentage increase in death above each corresponding vehicle control.

Figure 8

Modulation of PTEN function changes OSU-03012 lethality. (A) GBM14 cells were transfected with control plasmid (GFP) or a plasmid to express PTEN (PTEN-GFP). Twenty-four hours after transfection cells were treated with OSU-03012 (2 µM). Cells were isolated 24 and 48 h later for viability determination (n = 3, +/- SEM). Upper blots: portions of cells were isolated 24 and 48 h after OSU-03012 treatment and lysates subjected to blotting to determine the phosphorylation and expression of the indicated proteins. (B) GBM6 cells were transfected with control plasmid (shSCR) or a plasmid to knock down expression of PTEN (shPTEN). Twenty-four after transfection cells were treated with OSU-03012 (2 µM). Cells were isolated 24 and 48 h later for viability determination (n = 3, +/- SEM). Upper blots: portions of cells were isolated 24 and 48 h after OSU-03012 treatment and lysates subjected to blotting to determine the phosphorylation and expression of the indicated proteins.

Figure 9

OSU-03012 reduces the half-life of BiP/GRP78. (A) Upper: GBM12 cells were treated with vehicle or OSU-03012 (2 µM). Cell lysates were isolated at the indicated times to determine the expression of BiP/GRP78 and densitometry performed. Lower: GBM12 cells were treated with vehicle or OSU-03012 (2 µM). Total RNA was isolated at the indicated time points and quantitative RT-PCR performed to determine BiP/GRP78 levels (n = 3, +/- SEM). (B) GBM12 cells were treated with vehicle or cycloheximide (20 µg/ml), followed by OSU-03012 (2 µM). Cells were lysed to determine the protein expression of BiP/GRP78 compared with GAPDH and the relative intensity of protein levels presented graphically (n = 3, +/- SEM).

Figure 10

OSU-03012 prolongs animal survival using an orthotopic model and invasive primary human glioma cells. Upper: GBM6-luciferase cells (1 x 10⁶) were implanted into the brains of mice (45 total). Tumors were permitted to form for 16 d after which animals were grouped based on tumor luminosity. The panel shows that over the following 12 d treatment with 50 mg/kg OSU-03012 significantly reduced tumor mass (**p < 0.05) and interacted with radiation to further suppress tumor mass (#p < 0.05)