The tumor suppressor gene TUSC2 (FUS1) sensitizes NSCLC to the AKT inhibitor MK2206 in LKB1-dependent manner

Abstract

TUSC2-defective gene expression is detected in the majority of lung cancers and is associated with worse overall survival. We analyzed the effects of TUSC2 re-expression on tumor cell sensitivity to the AKT inhibitor, MK2206, and explored their mutual signaling connections, in vitro and in vivo. TUSC2 transient expression in three LKB1-defective non-small cell lung cancer (NSCLC) cell lines combined with MK2206 treatment resulted in increased repression of cell viability and colony formation, and increased apoptotic activity. In contrast, TUSC2 did not affect the response to MK2206 treatment for two LKB1-wild type NSCLC cell lines. In vivo, TUSC2 systemic delivery, by nanoparticle gene transfer, combined with MK2206 treatment markedly inhibited growth of tumors in a human LKB1-defective H322 lung cancer xenograft mouse model. Biochemical analysis showed that TUSC2 transient expression in LKB1-defective NSCLC cells significantly stimulated AMP-activated protein kinase (AMPK) phosphorylation and enzymatic activity. More importantly, AMPK gene knockdown abrogated TUSC2-MK2206 cooperation, as evidenced by reduced sensitivity to the combined treatment. Together, TUSC2 re-expression and MK2206 treatment was more effective in inhibiting the phosphorylation and kinase activities of AKT and mTOR proteins than either single agent alone. In conclusion, these findings support the hypothesis that TUSC2 expression status is a biological variable that potentiates MK2206 sensitivity in LKB1-defective NSCLC cells, and identifies the AMPK/AKT/mTOR signaling axis as an important regulator of this activity.

Figure 1. Endogenous and overexpressed levels of TUSC2 in LKB1-defective and wild type NSCLC cells.

Cells were transfected with DC-TUSC2 for 24 hours, and TUSC2 protein levels were detected by western blot with a rabbit anti-TUSC2 polyclonal antibody.

Figure 2. Inhibition of tumor cell viability and colony formation by TUSC2 transfection and MK2206 combined treatment in LKB1-defective and wild type NSCLC cells.

A) Forty-eight hours post-treatment, cells were assayed for viability as described in Materials and Methods. Cell viability was plotted against concentration of MK2206. B) Cells were transfected with DC-TUSC2. Twenty four hours post-transfection, cells were split, replated in triplicate, and grown in medium containing 400 µg/ml of the antibiotic G418 before treatment with 1 µM MK2206. Colonies were fixed with glutaraldehyde (6.0% v/v), stained with crystal violet (0.5% w/v), and counted using a stereomicroscope. Columns, mean of three different experiments, each with duplicate samples; bars, SD. *, P<0.05, compared with EV control; **, P<0.05, compared with EV+MK2206

Figure 3. Apoptosis mediated by TUSC2 transfection and MK2206 combined treatment involves caspase-9 activation.

Cells were transfected with DC-TUSC2 for 24 hours, starved for 24 hours and then treated with 1 µM MK2206 for 48 hours before measuring their apoptotic activity. A) DNA fragmentation was analyzed by flow cytometry, and relative apoptotic cells were calculated in terms of the FITC-positive values in cells. Columns, mean of three different experiments, each with duplicate samples; bars, SD. *, P<0.05, compared with EV control; **, P<0.05, compared with EV+MK2206; B) Proteins in cell lysates were separated with 12% SDS PAGE, probed with caspase-9 antibody to detect both full length caspase-9 (47 kDa) and large fragments of caspase-9 (35 kDa, 37 kDa). Immunoreactive bands were visualized with an Odyssey Imager.

Figure 4. Effective in vivo inhibition of tumor growth by TUSC2 systematic restoration and MK2206 combined treatment.

A subcutaneous mouse model of human NSCLC H322 was used to evaluate the combined effect of systemic delivery of the DC–based TUSC2 nanoparticles and MK2206 treatment on tumor growth inhibition. A) Tumor volume was calculated, taking length to be the longest diameter across the tumor and width to be the corresponding perpendicular diameter, using the following formula: length × width²×0.52. Tumor growth inhibition rate was calculated as 100%× (tumor size_treated/tumor size_control) on each measurement day. Bars, SD; B) Tumors were resected, fixed with 4% paraformaldehyde, paraffin-embedded for immunohistochemistry staining with the indicated antibodies, and examined under a Nikon TC200 fluorescence microscope equipped with a digital camera.

Figure 5. Stimulation of AMPK phosphorylation and kinase activity by TUSC2 in LKB1-defective cells.

A) HCC366, H322, and A549 were transfected with TUSC2 then either treated with 1 µM MK2206 for 24 hours or left untreated. AMPK kinase activity in the immunocomplexes was measured by phosphorylation of SAMS peptide as described in Materials and Methods. Columns, mean of three different experiments, each with duplicate samples; bars, SD. *, P<0.05, compared with EV control; **, P<0.05, compared with EV+MK2206. LKB1- defective cells HCC366 and H322 cells were co-transfected with 2 µg TUSC2 plasmid and 50 nM AMPK siRNA with Lipofectamine™ 2000. Twenty-four hours after transfection, cells were starved for 24 hours and treated with 1 µM MK2206 for an additional 48 hours. B) Cell lysis were collected for western blot analysis to assess levels of AMPK and p-AMPK proteins; or C) Cells were assayed for apoptosis as described in Materials and Methods. Columns, mean of three different experiments, each with duplicate samples; bars, SD. ^#, P<0.05, compared with TUSC2; ^##, P<0.05, compared with TUSC2+MK2206.

Figure 6. Inhibition of AKT and mTOR kinase activity by TUSC2 transfection and MK2206 combined treatment.

LKB1-defective HCC366, H322, and A549 were transfected with TUSC2 for 24 hours, starved for 24 hours and either treated with 1 µM MK2206 for 24 hours or left untreated. Cell lysates were collected for western blot analysis for the levels of A) p-AKT(S473) and p-AKT(Th308); and B) p-mTOR(S2448). AKT and mTOR was precipitated from 200 ug cell lysis using A) AKT or B) mTOR antibodies. The kinase activity of AKT and mTOR were measured with KLISA AKT and mTOR assay kit, respectively, using GSK-3α and S6K GST fusion proteins as substrates, respectively. Kinase activities were determined by ELISA, as substrate absorbance was measured at 450 nm, and reference wavelengths were measured at 540/595 nm using a Synergy 2 Multi-detection microplate reader. Columns, mean of three different experiments, each with duplicate samples; bars, SD. *, P<0.05, compared with EV control; **, P<0.05, compared with EV+MK2206.