Rab22a-NeoF1 fusion protein promotes osteosarcoma lung metastasis through its secretion into exosomes

It remains unknown for decades how some of the therapeutic fusion proteins positive in a small percentage of cancer cells account for patient outcome. Here, we report that osteosarcoma Rab22a-NeoF1 fusion protein, together with its binding partner PYK2, is sorted into exosomes by HSP90 via its KFERQ-like motif (RVLFLN142). The exosomal Rab22a-NeoF1 fusion protein facilitates the pulmonary pre-metastatic niche formation by recruiting bone marrow-derived macrophages. The exosomal PYK2 activates RhoA in its negative recipient osteosarcoma cells and induces signal transducer and activator of transcription 3 activation in its recipient macrophages to increase M2 phenotype. Consequently, lung metastases of its recipient osteosarcoma cells are promoted by this exosomal Rab22a-NeoF1 fusion protein, and this event can be targeted by disrupting its interaction with PYK2 using a designed internalizing RGD peptide.

(e, f) 12-36% (e) and 5%-40% (f) iodixanol density gradients centrifugation was performed using the exosomes derived from 143B cells stably expressing RAB22A-NeoF1 as described in "Materials and Methods" and followed by Western blotting.Data in e and f are representative of n = 3 biologically independent experiments.

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Figure.S1.The secreted RAB22A-NeoF1 fusion protein enhances metastasis in osteosarcoma.(a) Bioluminescence analyses of lung metastases from mice orthotopically injected with the indicated cells under treatment of either Vector-CM or RAB22A-NeoF1 CM. n = 5 biologically independent mice.Data are mean ± s.d.P values are shown.(b) 143B cells were co-cultured with the indicated stable cells for 5 days and then were subjected to migration and invasion assays.Data are mean ± s.d. of n = 3 biologically independent experiments.P values are shown.(c-f) Representative IVIS imaging (c), Bioluminescence analyses (d)

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Figure.S2.The RAB22A-NeoF1 fusion protein is present in exosomes to promote osteosarcoma metastasis.(a) Representative images of immunofluorescence staining for both intracellular RAB22A-NeoF1 and the indicated subcellular markers in U2OS cells stably expressing RAB22A-NeoF1.Scale bar, 5μm.
(g) Representative confocal microscopy image of U2OS cells treated with exosomes derived from 143B cells stably expressing Rab22a-NeoF1-EGFP (Rab22a-NeoF1-EGFP-exo) for 15min.The late endosomes (Red, denoted by RAB7) and GFP signals (Green) were shown.Scale bar, 5μm.(h) The U2OS cells were treated with or without exosomes derived from 143B cells stably expressing Rab22a-NeoF1-GFP (Rab22a-NeoF1-EGFP-exo) for 15 min, and subjected to flow cytometry analysis.(i) Bioluminescence analyses of lung metastases from mice orthotopically injected with the indicated cells under treatment of exosomes derived from the indicated stable ZOS-M cells.n = 5 biologically independent mice.Data are mean ± s.d.P values are shown.

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Figure.S3.RAB22A-NeoF1 fusion protein is sorted into exosomes via binding to HSP90 to promote osteosarcoma metastasis (a, b) The indicated stable 143B cells were lysed and analyzed by Western blotting.(c) The exosomes from the indicated stable 143B cells were purified and analyzed by Western blotting.The relative intensity of RAB22A-NeoF1 was quantified with ImageJ software and normalized with HSP70.Data in a-c are representative of n = 3 biologically independent experiments.
(d) The protein sequence of RAB22A-NeoF1.The yellow region indicates four and mutated amino acids are highlighted in red.(e)293T cells were co-transfected with the indicated plasmids and then were lysed and analyzed by immunoprecipitation using S protein beads and Western blotting.(f) The 143B cells stably expressing Rab22a-NeoF1 were treated with the HSP90 inhibitor (Ganetespib), HSP70 and HSC70 inhibitors (VER155008 and Apoptozole) for 24h, and then the exosomes were purified and analyzed by Western blotting.Data in e and f are representative of n = 3 biologically independent experiments.(g) Bioluminescence analysis of lung metastasis from mice orthotopically injected 143B-Luc cells with exosomes derived from the indicated stable 143B cells.n = 5 biologically independent mice.Data are mean ± s.d.P values are shown.

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Figure.S4.The functions of exosomes containing RAB22A-NeoF1 fusion protein on its negative recipient cancer cells requires its binding partner PYK2 from donor cells.(a) The heat maps of differential expression cluster in exosomes from the indicated stable 143B cells.(b) The protein compositions in exosomes from the indicated stable 143B cells.

Figure. S5 .
Figure.S5.The functions of exosomal RAB22A-NeoF1 fusion protein requires recruiting BMDMs to pulmonary pre-metastatic niche.(a) Mice were pre-educated with the indicated conditioned media (CM) for 3 weeks, and then were orthotopically or tail vein injected with 143B-Luc cells.The lung metastases were analyzed by Bioluminescence after 3 weeks.n =4 biologically independent mice.Data are mean ± s.d.P values are shown.(b) Mice orthotopically co-injected U2OS/MTX300-Luc cells with the indicated stable ZOS-M cells at the 10:1 ratio, and then intravenously injected liposome or PBS twice a week.The lung metastases were analyzed by bioluminescence.n = 5 biologically independent mice.Data are mean ± s.d.P values are shown.(c-f) Mice were pre-educated with the indicated exosomes combined with liposome or PBS for 3 weeks, and then were orthotopically injected with

Figure. S6 .
Figure.S6.The exosomal RAB22A-NeoF1 fusion protein promotes M2 polarization in its recipient macrophages via its binding partner PYK2.(a, b) Raw264.7 cells (a) and THP-1 cells (b) were incubated with the indicated exosomes for 24 h, and differential expression patterns of the M0, M1 and M2 phenotype markers were presented by a heatmap.

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Figure.S7.The 1-10aa of RAB22A-NeoF1 is responsible for its binding to PYK2.(a, b) 293T cells were co-transfected with the indicated plasmids and then were lysed and analyzed by immunoprecipitation using anti-Flag beads (a) or

Figure. S8 .
Figure.S8.Secretion of RAB22A-NeoFs proteins (a) The protein sequences of RAB22A-NeoF2-6.The yellow region indicates the KFERQ-like motif (aa52-57).(b) The exosomes derived from the indicated stable 143B cells were purified and subjected to Western blotting.Data are representative of n = 3 biologically independent experiments.

Figure. S9 .
Figure.S9.The interaction of RAB22A-NeoF1 fusion protein with PYK2, but not with smgGDS607, is dependent on its binding to HSP90.(a, b) 293T cells were co-transfected with the indicated plasmids and treated with the indicated HSP90 inhibitors for 24 h, and then were lysed and analyzed by immunoprecipitation using anti-Flag beads followed by Western blotting.(c)ZOS-M cells were treated with the indicated HSP90 inhibitors for 24 h and then were lysed and subject to immunoprecipitation using IgG and mAb RAD5-8 followed by Western blotting.Data in a-c are representative of n = 3 biologically independent experiments.