Acetylation dependent functions of Rab22a-NeoF1 Fusion Protein in Osteosarcoma

Abstract

Background: Rab22a-NeoF1 fusion gene containing the 1-38aa of Rab22a (Rab22a^1-38) 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 Rab22a^1-38.

Keywords: Rab22a-NeoF1; acetylation; metastasis; osteosarcoma; p300/CBP.

Figure 1

Rab22a-NeoF1 fusion protein is acetylated at lysine 7 (K7). (A) HEK293T cells transfected with Rab22a-NeoF1-SFB for 48 h, and cell lysates were subjected to immunoprecipitation (IP) with anti-Flag agarose, the IP complex was then analyzed by mass spectrometry. MS spectra of Ac-K containing the 5-ELKVCLLGDTGVGK-18 peptide obtained after trypsin digestion of the IP complex. (B, C) HEK293T cells transiently transfected with Rab22a-NeoF1-SFB or its K7R mutant for 24 h were treated with TSA (5 µM), NAM (5 mM), or both for 8 h. Cell lysates were subjected to IP with anti-Flag agarose, and then were analyzed by Western blotting. (D) ZOS-M cells were treated with both TSA (5 µM) and NAM (5 mM) for 24 h, cell lysates were subjected to IP using anti-K7ac-Rab22a-NeoF1 antibody, and then were analyzed by Western blotting using mAb RAD5-8.

Figure 2

The K7R mutant of Rab22a-NeoF1 lacks its capacities of enhancing migration, invasion and metastasis in vitro and in vivo. (A, B) Quantification analyses of migration and invasion assays (lower panel) using U2OS cells or U2OS/MTX300 cells stably expressing Vector, Rab22a-NeoF1 (WT) or its K7R mutant (K7R), as indicated. The expression level of WT and K7R Rab22a-NeoF1 in U2OS cells and U2OS/MTX300 cells were shown in the upper panel. (C-F) The orthotopic osteosarcoma metastasis model in vivo using the U2OS/MTX300-Luc cells stably expressing Vector, Rab22a-NeoF1 (WT) or its K7R mutant (K7R), as indicated. Representative images of mice (C). H&E staining of the lungs from representative tumor-bearing nude mice (D). Quantification analyses of Log₁₀(Average Radiance) of lung metastasis. (E) Quantification analyses of lung nodules (F). Quantification analyses of wet lung weight (G) from the nude mice used in C.

Figure 3

p300/CBP acetyltransferase are responsible for the K7 acetylation of Rab22a-NeoF1. (A,B) HEK293T cells were co-transfected Rab22a-NeoF1-SFB with the p300 (A) or CBP (B) specific siRNAs indicated acetyltransferases plasmids for 48 h, cell lysates were subjected to IP using anti-Flag agarose, and then were analyzed by Western blotting. (C) HEK293T cells transiently transfected Rab22a-NeoF1-SFB (WT) or its K7R mutant (K7R) with HA-CBP 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-S protein beads, and then were analyzed by Western blotting. (D) ZOS-M cells were transfected with HA-p300 or HA-CBP, cell lysates were subjected to IP using anti-K7ac-Rab22a-NeoF1 antibody, and then were analyzed by Western blotting by mAb RAD5-8. (E) Rab22a-NeoF1-WT-SFB or the Rab22a-NeoF1-K7R-SFB mutant was incubated with HA-CBP purified from HEK293T cells in acetylation buffer for 1 h. in vitro and then analyzed using anti-K7ac-Rab22a-NeoF1 antibody by Western blotting. (F) ZOS-M cells were lysed and subjected to IP using mAb RAD5-8, and then were analyzed by Western blotting using anti-p300, anti-CBP or hRAD5-8-v1-R5 antibody.

Figure 4

C646 inhibits migration, invasion and the lung metastases induced by Rab22a-NeoF1 in vitro and in vivo. (A, B) ZOS-M cells were treated with 5 µM C646 (A) or 5 mM salicylate (B) for 24 h, cell lysates were subjected to IP using anti-K7ac-Rab22a-NeoF1 antibody, and then were analyzed by Western blotting by mAb RAD5-8. (C, D) Quantification analyses of migration and invasion assays using U2OS cells (C) or U2OS/MTX300 (D) cells stably expressing Vector, Rab22a-NeoF1 (WT) or its K7R mutant (K7R), as indicated, with or without the treatment of 5 µM C646 for 24 h. (E-H) The orthotopic osteosarcoma metastasis model in vivo using the U2OS/MTX300-Luc cells stably expressing Vector, Rab22a-NeoF1 (WT) or its K7R mutant (K7R), as indicated. After cell injection into mice for 3 weeks, the mice were intraperitoneally injected with C646 (10 mg/kg/d) or the control (7.7% DMSO+40% PEG300+ddH2O) daily for 14 days. Lung metastasis were detected by in vivo fluorescent imaging two months after cell injection. Representative images of mice (E). H&E staining of the lungs from representative tumor-bearing nude mice (F). Quantification analyses of Log₁₀(Average Radiance) of lung metastasis (G). Quantification analyses of lung nodules (H). Quantification analyses of wet lung weight (I) from the nude mice used in E.

Figure 5

Rab22a-NeoF1 K7 deacetylation is regulated by HDAC6 and SIRT1. (A, B) HEK293T cells were co-transfected Rab22a-NeoF1-SFB with the indicated HA-tagged histone deacetylases plasmids for 48 h, cell lysates were subjected to IP using anti-Flag agarose, and were then analyzed by Western blotting. (C, D) ZOS-M cells were transfected with HA-SIRT1 (C) or HA-HDAC6 (D) for 48 h, cell lysates were subjected to IP using anti-K7ac-Rab22a-NeoF1 antibody, and then were analyzed by Western blotting by mAb RAD5-8. (E) ZOS cells were transfected with siRNA targeting SIRT1 and HDAC6 for 48 h, cell lysates were subjected to IP using anti-K7ac-Rab22a-NeoF1 antibody, and then were analyzed by Western blotting by mAb RAD5-8. (F) ZOS-M cells were lysed and subjected to IP using mAb RAD5-8, and then were analyzed by Western blotting using anti-SIRT1, anti-HDAC6 or hRAD5-8-v1-R5 antibody.

Figure 6

The inhibition of p300/CBP or the K7R mutant impairs the interaction of Rab22a-NeoF1 with SmgGDS607 and the activation of RhoA by Rab22a-NeoF1. (A) HEK293T cells were co-transfected SmgGDS607-HA with Rab22a-NeoF1-SFB (WT) or its K7R mutant (K7R), as indicated. Cell lysates were subjected to IP using anti-HA, and were then analyzed by Western blotting. (B) U2OS cells stably overexpressing Rab22a-NeoF1-SFB (WT) or its K7R mutant (K7R) were treated with 10 μM C464 for 24 h, as indicated, cell lysates were subjected to IP using mAb RAD5-8 and/or Western blotting using hRAD5-8-v1-R5 antibody. (C) U2OS/MTX300 cells stably overexpressing Vector, Rab22a-NeoF1-SFB (WT) or its K7R mutant (K7R) were subjected to the RhoA GTPase activation assay.

Figure 7

The proposed model for the K7 acetylation of Rab22a-NeoF1. Acetylation of Rab22a-NeoF1 at K7, which is acetylated by p300/CBP and deacetylated by both SIRT1 and HDAC6, may be required for its association with SmgGDS607, resulting in more RhoA-GTPase to promote lung metastasis. C646 or salicylate targeting p300/CBP may be benefit for the osteosarcoma patients who are positive for Rab22a-NeoF1.