N6-Methyladenosine Modification of ANLN Enhances Hepatocellular Carcinoma Bone Metastasis

Bones are categorized as the second most prevalent location of extra-hepatic metastasis in Hepatocellular Carcinoma (HCC), which is linked to an extremely poor prognosis due to limited therapeutic options. N6-methyladenosine (m6A) is a prominent modification involved in HCC, but the exact mechanisms on how m6A modifications induce HCC bone metastases (BM) remain unclear. The key modulators responsible for the abundant m6A RNA modification-induced HCC BM was found to be the METTL3 and YTHDF1. The expression of Anillin actin-binding protein (ANLN) was dramatically higher in HCC with BM tissues, and its messenger RNA (mRNA) stability was enhanced via m6A epitranscriptomic regulation by METTL3 and YTHDF1. High METTL3 and YTHDF1 expression along with nuclear ANLN protein was clinically correlated with BM in HCC patients. Furthermore, HCC BM was attributed to over-expression of nuclear ANLN forming a transcriptional complex with SP1 which enhanced KIF2C transcriptional activity to activate the mTORC1 pathway, therefore increased the expression of RANKL and disproportionated RANKL-OPG expression in bone microenvironment leading to malignant neoplasms invade bone tissue. In addition, inhibition of ANLN m6A modification by DZNeP attenuated HCC BM. This data provides meaningful understanding of the modulation and association of m6A epitranscriptomic-regulated BM in HCC, and moreover, defines potentially valuable therapeutic targets.

Methylated RNA immunoprecipitation-qPCR was performed using an adaptation of a published method [3]. After denaturation of poly-A-purified RNA for 10 min at 70 °C, it was immediately cooled on ice and treated with an anti-m 6

Enzyme-linked immunosorbent assay
The levels of RANKL and OPG in culture supernatants obtained over 48 h were measured using the Human RANKL and OPG kits (both Ray Biotech), following the provided directions.

Osteoclast differentiation assays
Bone marrow cells (BMCs) were obtained from the femurs of four-week-old mice and grown in minimum essential medium (MEM) with 15% FBS and macrophage colonystimulating factor (M-CSF) (50 ng/mL) for three days. Adherent cells were harvested, washed three times with PBS, and plated in 12-well plates with conditioned medium from treated HCC cells, M-CSF (25 ng/mL), and RANKL (100 ng/mL) for three days.
The cells were stained using a tartrate-resistant acid phosphatase positive (TRAP) kit (Sigma Aldrich), according to directions, to analyze osteoclast and preosteoclast formation. TRAP-stained cells with a minimum of three nuclei were defined as osteoclasts. A minimum of nine fields per plate were evaluated and the mean osteoclast numbers were recorded.

Animal models
All the mouse experiments which were performed according to the Naval Medical University's approved guidelines. The investigation complied with all applicable ethical regulations related to animal research. To investigate the influence of ANLN on HCC cell lines' BM ability, luciferase-labelled Huh7 (1 × 10 7) cells in 50 µL 1× PBS were injected into the left ventricles of four-to six-week-old female nude using a 100-µl Hamilton Microliter syringe as described previously [5]. The BMs were monitored by the IVIS@

RNA immunoprecipitation (RIP) assays
RIP assays were performed using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (17-700, Millipore), according to provided directions.
Magnetic beads were coated with 5 μg of the appropriate antibodies and incubated with cell lysates overnight at 4 °C. After six washes, the complexed material was treated with proteinase K digestion buffer, followed by extraction of the RNA with phenolchloroform. Interactions between YTHDF1 and ANLN mRNA were assessed qPCR and expressed relative to the input. Details of the antibodies are provided in Supplementary   Table 9.

Chromatin Immunoprecipitation (ChIP)
The Magna ChIP G Kit (17-611; Millipore) was used in accordance with the manufacturer's directions. After cross-linking of cells with 37% formaldehyde (Sigma F8775) solution (final concentration 1%) for 10 min and quenching with glycine, the PBS-washed cells were resuspended in lysis buffer with protease inhibitors and vortexed intermittently for 15 min at 4 °C. After centrifugation (800 g; 5 min), the pelleted cells were resuspended in nuclei lysis buffer and sonicated with a Misonix S-4000 sonicator at 50% amplitude 15-s pulse with 90-s rest between each pulse (10 cycles). The size of the chromatin was assessed by electrophoresis and was immunoprecipitated with either 3 μg anti-TCF4 or 3 μg anti-IgG (Millipore 17-10109) overnight at 4 °C. ChIP fragments were examined using RT-PCR and the primers provided in Supplementary Table 11.

Luciferase activity report assay
Wild-type (WT) and mutant (Mut) ANLN bind peaks (shown in Fig.5H) were inserted into pGL3 luciferase reporter by BgIII digestion. Huh7 and MHCC-97H cells were seeded in 48-well plates and the adherent cells were co-transfected with pGL3-binding peak-WT and-Mut plasmids together with Renilla using Lipofectamine™ 2000 (Invitrogen) for 48 h. Firefly and Renilla activities were measured and firefly activity was normalized to that of Renilla.

Immunoprecipitation (IP) and LC-MS
The Dynabeads Co-Immunoprecipitation Kit (14321D, Thermo Fisher) was used for IP, following the provided directions. Proteins were separated on SDS-PAGE and the bands were digested with trypsin. The peptides were applied to an LC gradient for 2 h and were analyzed on a Q Exactive HF mass spectrometer (Thermo Fisher Scientific).
The data were compared with the mouse database by MaxQuant (https://maxquant.org/).
The intensity-based absolute-protein-quantification (iBAQ) intensity was used for inter-sample comparisons. The antibodies used are shown in Supplementary Table 9.

Western blotting (WB)
Cells were lysed in RIPA buffer with protease and phosphatase inhibitors, as well as DTT and benzonase (Sigma-Aldrich). Protein concentrations were measured using a BCA kit (Thermo Scientific Pierce, Loughborough, UK). WB was performed as previously described using NuPAGE@Novex 4-12% Bis-Tris protein gels (Novex, Life Technologies, Carlsbad, CA, USA). The antibodies used are shown in Supplementary Table 9.

Nuclear and cytoplasmic protein extraction
Cells were fractionated using a cytoplasmic and nuclear protein/RNA extraction kit (Thermo Fisher Scientific) according to the supplied protocol. The loading controls for the nuclear and cytoplasmic fractions were laminB1 and α-tubulin, respectively.

Immunofluorescence
Cells (4x10 4 ) were grown in confocal plates, fixed with 4% formaldehyde for 20 min, and permeabilized with 0.1% Triton X-100 for 20 min. The cells were blocked with 5% BSA in Tris-buffered saline with Tween 20 TBST) and were probed with the appropriate primary antibodies overnight at 4 °C. After incubation with secondary antibodies (30 min, RT) and nuclear staining with DAPI, the cells were examined and imaged with a confocal microscope (Olympus, Tokyo, Japan). The antibodies used are shown in Supplementary Table 9.

Patient-Derived Xenografts
For the patient-derived tumor xenograft (PDX) model, fresh tumor tissues were cut into 3x3 cm sections and implanted subcutaneously into the mouse flanks. The mice were given 3-deazaneplanocin A (DZNeP; 8 mg/kg per mouse) or dimethylsulfoxide (DMSO) orally 7 times for 2 weeks. The growth of the tumors was assessed at the indicated times. This procedure was approved by the EHBH Ethics Committee. Growth rates were calculated as previously described [6].

m 6 A-Seq
The m 6 A-Seq method was used to measure m 6 A RNA methylation. The library was constructed using the Next® Ultra™ II RNA Library Prep Kit (E7775; NEB), quantified with a Qubit Fluorometer (Thermo Fisher Scientific), and size distributions confirmed with Tape Station D1000 Screen Tape (Agilent Technologies). Sequencing was done on an Illumina platform using PE150 (Shanghai OE Biotech, Shanghai, China).

CUT&Tag library and sequencing
Huh7 cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and grown according to the supplier's protocol. We used the following antibodies: ANLN Primary antibody. A cleavage under targets and tagmentation (CUT&Tag) library was constructed using Hyperactive TM In-Situ ChIP Library Prep Kit (TD901, Vazyme, Nanjing, China). In short, after harvesting, counting, and centrifugation (600 g; 3 min; RT), 60-500 000 cells aliquots were washed twice in 500 μL wash buffer by gentle pipetting. Ten microliters of prepared Concanavalin Acoated magnetic beads were added to each sample and incubated at RT for 10 min.
Following the protocol, the cells were subjected to successive incubations with the beads, primary antibody, secondary antibody, and Hyperactive PG-TN5/PA-TN5 Transposon, and then fragmented. The fragmented DNA was isolated and amplified by PCR to obtain the library, which was sequenced using PE150 on the Illumina sequencer (Shanghai OE Biotech, China).

Data processing for CUT&Tag
We used Fastp v0.20.0 was used to remove adapter and low-quality reads.