GALNT12 suppresses the bone-specific prostate cancer metastasis by activating BMP pathway via the O-glycosylation of BMPR1A

Bone metastasis caused the majority death of prostate cancer (PCa) but the mechanism remains poorly understood. In this present study, we show that polypeptide N-acetylgalactosaminyltransferase 12 (GALNT12) suppresses bone-specific metastasis of PCa. GALNT12 suppresses proliferation, migration, invasion and cell division ability of PCa cells by activating the BMP pathway. Mechanistic investigations showed that GALNT12 augments the O-glycosylation of BMPR1A then actives the BMP pathway. Activated BMP signaling inhibits the expression of integrin αVβ3 to reduce the bone-specific seeding of PCa cells. Furthermore, activated BMP signaling remolds the immune microenvironment by suppressing the STAT3 pathway. Our results of this study illustrate the role and mechanism of GALNT12 in the process of bone metastasis of PCa and identify GALNT12 as a potential therapeutic target for metastatic PCa.

The human GALNT12 mutant plasmid, mouse GALNT12 and its mutant plasmid and BMPR1A mutant plasmid were constructed by YouBio (Hunan, China).Lipofectamine 2000 was used for plasmids transfection according to the instruction.
To construct stably expressed cells, target cells were infected with lentivirus for 12 h and selected with antibiotic for 4 days.

RNA sequencing and quantitative real-time PCR (qRT-PCR) analysis
Total RNA was extracted from RM1 parental , RM1 LuM3 and RM1 BM4c cells during the logarithmic growth phase.Firstly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads.Fragmentation was carried out using divalent cations under elevated temperature in an Illumina proprietary fragmentation buffer.First strand cDNA was synthesized using random oligonucleotides and Super Script II.Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities and the enzymes were removed.After adenylation of the 3′ ends of the DNA fragments, Illumina PE adapter oligonucleotides were ligated to prepare for hybridization.To select cDNA fragments of the preferred 400-500 bp in length, the library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, CA, USA).DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 15 cycle PCR reaction.Products were purified (AMPure XP system) and quantified using the Agilent high sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent).The sequencing library was then sequenced on NovaSeq 6000 platform (Illumina) Shanghai Personal Biotechnology Cp. Ltd.
qRT-PCR was performed as described previously [1].Briefly, the total RNA was extracted from cells using TRIzol reagent (Invitrogen, Waltham, USA) and reversely transcribed to cDNA using RT SuperMix (Vazyme, Nanjing, China).qRT-PCR was performed on QuantStudio™ 6 Flex System (Applied Biosystems, Foster City, CA) using SYBR Master Mix (Vazyme, Nanjing, China).The relative expression of mRNA was normalized to ACTB by the 2 -ΔCtΔCt method and the primers purchased from Generay (Shanghai, China) were listed in Table S1.
For H&E staining, slides were stained in hematoxylin for 1 min and eosin for1 min.All the slides were viewed by a light microscope (Leica DM1000, Leica Biosystems Inc., Heerbrugg, GER).

Western blot (WB) analysis
WB was performed as described previously [1].Briefly, cells were lysed by RIPA buffer (Byeotime, Shanghai, China) which contains phosphatase inhibitor and protease inhibitor and the lysate was centrifuged at 12,000 g for 10 min.The supernatant was transferred into a new tube and boiled at 95 ℃ with loading buffer for 5 min.ACTB was selected as the reference protein.The information of antibodies used in this study was list in Table S2.

MTT assay
MTT assay was performed to evaluate the proliferation of PCa cells.3,000 cells were seeded into 96-well plates.10 μL MTT solution was added to the wells at different time points.After incubation for 2 h, the supernatant was removed.100 μL DMSO was added to the wells to dissolve the purple formazan crystals subsequently and the plate was placed in a 37 ℃ environment for 30 min.Absorbance was measured at 490 nm, using a microplate reader (Tecan Infinite, M annedorf, Switzerland), absorbance 630 nm was selected as a reference.

Transwell migration and invasion assay
For migration assay, 1×10 5 cells in 200 μL FBS-free medium were added to the upper transwell chamber (8 μm, BIOFIL, Guangzhou, China) and incubated at 37 ℃.After 24 h, the chamber was fixed with 4% paraformaldehyde (PFA) and stained with the crystal violet and the cells on the bottom of chamber were counted under a microscope.
For invasion assay, 100 μL diluted matrigel (Corning, NY, USA) was spread on the bottom of upper transwell chamber and placed in 37 ℃ for 4 h and the following procedures were the same as migration assay.

Wound healing assay
5×10 5 cells were seeded into a 6-well plate per well.When the density of cells reached about 90% confluence, an artificial wound was scratched with a 1 mL pipette tip.The floating cells were removed by gently washing with PBS for several times and FBSfree medium were added to the wells subsequently.Images were taken at 0h and appropriate end time to estimate migration abilities of cells.
Spheres more than 50 μm were calculated and photographed.

Clone formation assay
100 cells were inoculated into 12-well plate and cultured with complete medium for 10-14 days.Cells were then fixed with 4% PFA for 15 min and stained with the crystal violet for 10 min.The number of clones formed more than 50 cells was calculated

Cell cycle, apoptosis and anoikis assay
Cells during the logarithmic growth phase were collected and fixed with 75% ethanol in 4 ℃ overnight for cell cycle assay.Then cells were washed with PBS twice and then incubated with propidine iodide (PI) for 30 min and then tested by a flow cytometry (ACEA, Hangzhou, China).
For apoptosis assay, cells were cultured in FBS-free medium for 24h and then collected and washed with PBS twice.Cells were stained with Annexin V-FITC and PI (Vazyme, Nanjing, China) for 10 min.The apoptosis was tested by the flow cytometry.
For anoikis assay, 5×10 5 cells were seeded into ultra-low attachment plates and cultured with complete medium for 24h.The apoptosis of cells was tested by the flow cytometry with the Apoptosis Kit (Vazyme, Nanjing, China).and RM BM4c cells compared to RM1 parental cells.C: Heat map analysis showing the genes expression between RM1 parental , RM1 LuM3 and RM BM4c cells.D: KEGG enrichment analysis of differential expressing genes in RM1 LuM3 and RM BM4c cells compared to RM1 parental cells.E: GO enrichment analysis of differential expressing genes in RM1 LuM3 and RM BM4c cells compared to RM1 parental cells.

Figure
Figure S1.A: PCA analysis showing the profile of RM1 parental , RM1 LuM3 and RM BM4c cells.B: Volcano map analysis showing the differential expressing genes in RM1 LuM3