Validation of SAG/RBX2/ROC2 E3 ubiquitin ligase as an anticancer and radiosensitizing target

Abstract

Purpose: Sensitive to apoptosis gene (SAG; also known as RBX2 or ROC2) was originally cloned as a redox-inducible antioxidant protein and was later characterized as a RING component of SCF E3 ubiquitin ligases. SAG overexpression inhibits apoptosis induced by many stimuli both in vitro and in vivo. SAG mRNA was overexpressed in human lung tumor tissues with a correlation to poor patient survival. To investigate whether SAG serves as an anticancer target, we determined the effect of SAG silencing on cell proliferation, survival, and radiosensitivity.

Experimental design: SAG protein expression in human tumors was evaluated by immunohistochemical staining using tumor tissue arrays. SAG expression in cancer cells was knocked down by siRNA silencing. The anticancer effects of SAG silencing were evaluated by in vitro assays for cell growth and survival and by an in vivo orthotopic xenograft tumor model. Radiosensitization by SAG silencing of human cancer cells was determined by clonogenic survival assay. Apoptosis induction was evaluated by fluorescence-activated cell sorting analysis, caspase-3 activation assay, and Western blotting of apoptosis-associated proteins.

Results: SAG was overexpressed in multiple human tumor tissues compared with their normal counterparts. SAG silencing selectively inhibited cancer cell proliferation, suppressed in vivo tumor growth, and sensitized radiation-resistant cancer cells to radiation. Mechanistically, SAG silencing induced apoptosis with accumulation of NOXA, whereas SAG overexpression reduced NOXA levels and shortened NOXA protein half-life.

Conclusions: The findings showed that SAG E3 ubiquitin ligase plays an essential role in cancer cell proliferation and tumor growth and may serve as a promising anticancer and radiosensitizing target.

Figure 1. The expression of SAG in human tumors and normal counterparts

(A) SAG overexpression in multiple human primary tumor tissues. Tumor tissue arrays containing multiple normal and tumor tissues from different organs were stained with purified SAG monoclonal antibody on the DAKO AutoStainer using the DakoCytomation EnVision+ System-HRP (DAB) detection kit, and counterstained with hematoxylin (Surgipath). The stained slides were observed under microscope (OLYMPUS 1X71) and images were acquired using software DP controller. (B) SAG staining in lung tissues, normal vs. cancer. Lung tumor tissue arrays containing normal lung and tumor tissues were stained for SAG expression. Stained normal and tumor tissues were classified into four groups (+ to ++++) according to staining intensity of each tissue. (C) The percentage of normal or tumor tissues in each staining group. Tissue samples with different staining intensity were grouped and tabulated.

Figure 2. SAG silencing selectively inhibits the growth of human cancer cells

H1299 human lung cancer cells, U87 human glioblastoma cells, NL20 normal bronchial epithelial cells and MRC-5 lung fibroblast cells were infected with LT-CONT and LT-SAG for 96 hrs, and then split for assays as follows. (A) SAG silencing effects, determined by immunobloting (IB) with β-actin as the loading control 96h post cells splitting. (B) ATPlite cell proliferation assay. Cells after lentivirus-based siRNA silencing were split and seeded into 96-well plates with 3000 cells per well in quadruplicates and subject to ATPlite cell proliferation assay over periods up to 96 hrs. *P<0.05; **P<0.01. (C) Clonogenic cell survival assay in H1299 (top panel) and U87 (bottom) cells. Cells after lentivirus-based siRNA silencing were split, seeded into 6-well plates with 100 cells (H1299) or 300 cells (U87) per well in triplicates, and incubated at 37 °C for 9 days, followed by 0.05% methylene blue staining and colony counting. (D) Soft agar anchorage-independent growth assay in H1299 and U87 cells. Ten thousand cells after lentivirus-based siRNA silencing were seeded in 0.33% agar containing 1× cell culture medium and 10% FBS in 60-mm petri dish, and grown at 37°C for 14 days, followed by staining with p-iodonitrotetrazolium overnight and colony counting.

Figure 3. SAG silencing sensitizes cancer cells to radiation

Cells after lentivirus-based siRNA silencing were seeded in six-well plates at three different cell densities in duplicates. The next day, cells were exposed to different doses of radiation followed by incubation at 37°C for 9 days for colony counting. The surviving fraction was calculated and plotted after comparison with 0 Gy corresponding controls. The sensitizing enhancement ratio (SER) was calculated as the ratio of the inactivation dose under scrambled siRNA control conditions divided by inactivation dose after SAG silencing. Shown is value ±SEM from three independent experiments.

Figure 4. SAG silencing induces apoptosis with Noxa accumulation

H1299 and U87 cells were infected with LT-SAG, along with LT-CONT for 96 hours, then split and cultured for 72 hours, followed by PI staining and FACS analysis for apoptosis detection, caspase-3 activity assay, cell cycle profile and Western blotting for the levels of apoptosis-associated proteins. (A) Induction of apoptosis by SAG silencing. Apoptotic cells were determined by sub-G1 fraction in FACS analysis; (B) Caspase-3 activation upon SAG silencing. Caspase-3 activity in infected cells was determined by Caspase-3 activity assay. For A and B, shown is value ±SEM of three independent experiments. (C) The expression of apoptosis-associated proteins. The status of a panel of apoptosis-associated proteins including pro-apoptosis proteins and anti-apoptosis proteins were detected by Western blotting with β-Actin as the loading control.

Figure 5. SAG manipulation changes Noxa level and protein half-life

(A) SAG overexpression eliminates endogenous Noxa. U87 cells were transiently transfected with pcDNA3-FLAG-SAG, along with pcDNA3 as a control. Cells were harvested 30 hr later and subjected to Western blotting using anti-Noxa antibody with β-Actin as the loading control. (B) SAG overexpression reduced the level of ectopically overexpressed Noxa. U87 cells were transiently cotransfected with FLAG -Noxa and FLAG-SAG or FLAG -Noxa and pcDNA3. Cells were harvested 30 hr later and subjected to Western blotting using anti-FLAG antibody (For FLAG-Noxa) and anti-SAG (for FLAG-SAG and endogenous SAG) with β-Actin as the loading control. (C) SAG overexpression shortened the protein half life of Noxa. U87 cells were transiently cotransfected with FLAG -Noxa and FLAG-SAG or FLAG -Noxa and pcDNA3. Twenty-four hrs later, cells were treated with CHX at 20 μg/ml for indicated periods of time, followed by Western blotting using antibodies against FLAG (for FLAG-Noxa), SAG (for FLAG-SAG) with β-Actin as the loading control. The relative Noxa levels were quantified by densitometry analysis using ImageJ1.410 image processing software. (D) SAG knockdown extended the half life of endogenous Noxa. U87 cells were infected with LT-SAG, along with LT-CONT for 96 hrs and then split. Twenty-four hrs later, cells were treated with CHX at 20 μg/ml for indicated periods of time, followed by Western blotting using antibody against endogenous Noxa with β-Actin as the loading control. The relative Noxa levels were quantified by densitometry analysis using ImageJ1.410 image processing software. Endo-Noxa: endogenous Noxa.

Figure 6. SAG silencing inhibits the growth of orthotopic pancreatic tumors

PANC-1 human pancreatic carcinoma cells were infected with LT-CONT and LT-SAG, followed by the determination of SAG silencing effect (A), cell survival assay in vitro (B), tumor formation in vivo (C), and Western blotting (D). (A) SAG silencing effects. Panc-1 cells stably transfected with luciferase were infected with LT-CONT or LT-SAG, and the SAG levels were determined 96 hrs post infection by Western blotting with β-actin as the loading control. (B) Clonogenic cell survival assay. Cells after SAG silencing were split, seeded into 6-well plates with 100 cells per well in triplicates, and incubated at 37 °C for 9 days, followed by 0.05% methylene blue staining and colony counting. (C) Bioluminescence imaging of implanted tumors and measurement of tumor weight. Panc-1 cells stably transfected with luciferase were infected with LT-CONT or LT-SAG and implanted into pancreases of the mice (5 mice per group) for the evaluation of tumor growth in vivo. After 5 weeks, tumors were bioluminescence imaged using a cryogenically cooled imaging system coupled to a data acquisition computer running Living Image software at the University of Michigan Small Animal Imaging Core. Mice were then sacrificed, tumors harvested and weighed. (D) NOXA expression in tumors. The level of Noxa, SAG in tumor tissues was determined by Western blotting using antibodies against Noxa and SAG with β-Actin as the loading control.