Acetylation dependent functions of Rab22a-NeoF1 Fusion Protein in Osteosarcoma

Background: Rab22a-NeoF1 fusion gene containing the 1-38aa of Rab22a (Rab22a1-38) plays a decisive role in driving tumor metastasis by activating RhoA via binding to SmgGDS607. However, its intercellular regulation remains unknown. Methods: The Lys7 (K7) acetylation of Rab22a-NeoF1 was initially identified by mass spectrum. Co-transfection, immunoprecipitation and Western blotting were used to characterize the acetyltransferases and deacetylases responsible for the K7 acetylation of Rab22a-NeoF1, and to define the interaction of proteins. The specificity of K7 acetylation of Rab22a-NeoF1 was determined by its specific anti-K7ac-Rab22a-NeoF1 antibody and its K7R mutant. RhoA-GTP was measured by RhoA activation assay. The migration and invasion were assessed by Transwell assay without and with Matrigel matrix, respectively. The orthotopic osteosarcoma metastasis model in vivo was used to monitor the lung metastases of U2OS/MTX300-Luc stably expressing Vector, Rab22a-NeoF1 or its K7R mutant with or without C646, a relatively specific inhibitor of p300/CBP. The unpaired Student t test was used for the statistical significance. Results: The K7 of Rab22a-NeoF1 is acetylated by p300/CBP while is de-acetylated by both HDAC6 and SIRT1. The K7R mutant of Rab22a-NeoF1 lacks its binding to SmgGDS607 and subsequently lost its promoting functions, such as activation of RhoA, cell migration, invasion and lung metastasis in osteosarcoma in vitro and in vivo, which are also diminished by p300/CBP inhibitor C646. Conclusion:The promoting function of Rab22a-NeoF1 is dependent on its K7 acetylation in osteosarcoma, and targeting this acetylation (e.g., C646) may benefit cancer patients, in particular osteosarcoma patients, who are positive for the Rab22a1-38.

(A, B) HEK293T cells transfected with SFB-tagged Rab22a-NeoF1 for 48 h, and cell lysates were subjected to immunoprecipitation (IP) using anti-Flag agarose, the IP complex was then analyzed by mass spectrometry. MS spectra of K-GG (A), K-dimethyl (B) containing the 5-ELKVCLLGDTGVGK-18 peptide obtained after trypsin digestion of the IP complex.
(C-E) HEK293T cells were transiently transfected with Rab22a-NeoF1-SFB (WT) or its K7A mutant (K7A), as well as Myc-p53 that was used as the positive control for methylation. After 48 h, cell lysates were subjected to IP using anti-S protein beads or anti-c-Myc agarose, and then were analyzed by Western blotting using mono-methyl (C), di-methyl (D) or tri-methyl (E) lysine antibody. (F) HEK293T cells transiently transfected Rab22a-NeoF1-SFB (WT) or its K7R mutant (K7R) with HA-Ub for 24 h were treated with both TSA (5 μM) and NAM (5 mM) for 8 h. Cell lysates were subjected to IP using anti-Flag agarose, and then were analyzed by Western blotting.   Cell lysates were subjected to IP using anti-HA or anti-S protein beads, and then were analyzed by Western blotting. (E) ZOS-M cells were treated with both TSA (5 μM) and NAM (5 mM) for 8 h. Cell lysates were subjected to IP using mAb RAD5-8, and then were analyzed by Western blotting using anti-HDAC6, anti-SIRT1 or hRAD5-8-v1-R5 antibody.

Generation of stable cell lines
Cells were infected by lentivirus supernatant in the presence of 8 mg/ml Polybrene for 18-24 h. 48 h after infection, the cells were selected using cell medium containing 0.5 mg/ml puromycin.

Immunoblotting and immunoprecipitation
For Western blotting, cells were lysed in RIPA lysis buffer containing protease inhibitor and phosphatase inhibitor cocktails. Lysates were cleared by centrifugation at 12,000 rpm for 20 min at 4°C. For immunoprecipitation, the lysate were first incubated with anti-Flag agarose or anti-S-protein beads overnight at 4°C, and then the precipitates were washed five times with cold RIPA buffer and were eluted with 5 X SDS-PAGE. After SDS-PAGE, the proteins were transfered from the gel to the membrane. The membrane was blocked in PBST with 5% nonfat milk for 1-4 h in room temperature. Incubate the membrane with appropriate dilutions of primary antibody in antibody dilution buffer overnight at 4°C. Wash the membrane with PBST for three times. Incubate the membrane with the recommended dilution of conjugated secondary antibody in 5% nonfat milk blocking buffer at room temperature for 1 h. Wash the membrane with PBST for three times. Acquire image using darkroom development techniques for chemiluminescence, or normal image scanning methods for colorimetric detection.

Mass spectrometric analysis
HEK293T cells transfected with SFB-tagged Rab22a-NeoF1 expression plasmids for 48 h were collected and lysed with RIPA buffer, and the cell lysates were immunoprecipitated with anti-Flag agarose beads. The Flag peptide-eluted material was resolved by 10% SDS-PAGE. The Rab22a-NeoF1 bands were excised from the gel and were subjected to tryptic digestion and mass spectrometry. Protein and its modification were identified through the database search, and peptide identifications were validated with Peptide Prophet.

Transwell assay
The migration and invasion assays of osteosarcoma cells were performed using 24 wells

Cell viability assay
U2OS, or U2OS/MTX300 cells were seeded in 96-well plates at a density of 6,000 cells per well. Cells were then treated with different concentrations of C646 (0, 2.5, 5, 10, or 20 μM) or SA (0, 1, 5, 10, or 20 μM) for the indicated times, and the cell viability was measured by MTT assay.

RhoA GTPase activation assay
RhoA GTPase activation assay was performed with RhoA Pull-down Activation Assay Biochem Kit (bead pull-down format) following the direction of the manufacturer's protocol.

In vivo studies in mice
The study is compliant with all relevant ethical regulations regarding animal research. Animal experiments were approved by the Animal Research Committee of Sun Yat-sen University Cancer Center and performed in accordance with established guidelines. U2OS/MTX300luc cells stably overexpressing Vector, Rab22a-NeoF1 or its K7R mutant were prepared, and nude mice were purchased from Beijing Vital River Laboratory Animal Technology.
1×10 6 cells in PBS with 1% FBS were injected into distal femur, proximal tibia of each nude mouse (10 mice per group). After two months, lung metastases of U2OS/MTX300-luc cells were measured by in vivo fluorescent imaging and all mice were sacrificed and lungs with metastasis were harvested, and wet lungs were weighted and lung metastasis nodes were counted. For the treatment of C646, which was dissolved in ddH2O with 7.7% DMSO and 40% PEG300 at daily dose of 10 mg/kg for 14 days, was intraperitoneally injected into mice after the injection of U2OS/MTX300-luc cells for 3 weeks.

Statistical analysis
All experiments were performed at least three times. Data were analyzed with GraphPad Prism v.8 (GraphPad) and SPSS statistics. Student's t-test was used to compare the differences between two groups. *p<0.05 was considered as statistically significant while **p<0.01, ***p<0.001, ****p<0.0001 as highly significant.