2.1 Cell culture

The human head and neck squamous cell carcinoma (HNSC) cell line FADU, as well as the breast cancer cell lines MCF7 and MDA-MD-231, were acquired from the American Type Culture Collection (Manassas, VA, USA). These cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum at 37 °C and 5% CO2. For hypoxia induction, cells were maintained in 1% O2, 5% CO2, and 94% N2 for 18 h.

2.2 Plasmids and transfection

Plasmids pHA-HIF-1α, pHA-HIF-1α (ΔODD), and pHA-HIF-1α (LCLL), which express wild-type, constitutively active, and inactive HIF-1α, respectively, were kindly provided by Dr. L. E. Huang (University of Utah, Salt Lake City, UT, USA) [14]. Expression constructs Myc-DDK-tagged ALKBH4 and pCMV6-Entry vector were obtained from Origene (Rockville, MD, USA). The ALKBH4 promoter, with either wild-type or mutant hypoxia response elements (HRE) (pGL3-ALKBH4-HRE and pGL3-ALKBH4-mut 1), was subcloned into the pGL3-Basic vector in front of the luciferase gene for the reporter assay. For RNAi-mediated knockdown, the target sequences of HIF-1α (5′-GTGATGAAAGAATTACCGAAT-3′), ALKBH4 (5′-GTGATGCTGATCGAGGACTTT-3′), and scrambled control (5′-CCTAAGGTTAAGTCGCCCTCG-3′) were constructed into pLKO.1-puro vectors. For transfection, cells seeded for overnight were transfected using Lipofectamine 2000 (Thermo Fisher Scientific) following the manufacturer’s instructions. To generate stable cell lines, cells were transfected with plasmids for 48 h and selected with optimal antibiotics for two weeks.

2.3 Quantitative real-time PCR and Western blot analysis

As previously described [15], cells were harvested for RNA isolation using TRIzol reagent. Subsequently, cDNA was synthesized employing the MultiScribe Reverse Transcriptase system (Thermo Fisher Scientific) following the manufacturer’s instructions. For the quantification of mRNA expression levels, we performed quantitative real-time PCR using Fast SYBR Green Master Mix (Thermo Fisher Scientific) along with specific primers (Table S1). For Western blot analysis, whole-cell extracts were prepared, quantified, and boiled, as previously described [16]. Samples were subjected to analysis using SDS-PAGE, and protein were probed with antibodies against ALKBH4 (Novus Biologicals, Inc. Littleton, USA), HIF-1α, N-cadherin (BD Biosciences, Bedford, MA, USA), E-cadherin (Cell Signaling Technology, Danvers, MA, USA), vimentin, Flag (Sigma-Aldrich, St Louis, MO, USA), plakoglobin (Abcam, Cambridge, UK), GLUT1 (Millipore, Burlington, MA, USA), VEGF (Thermo Fisher Scientific), and β-actin (Genetex, Alton Pkwy Irvine, CA, USA).

2.4 Luciferase reporter and chromatin immunoprecipitation (ChIP) assays

FADU and MCF7 cells were co-transfected with reporter plasmids containing the ALKBH4 promoter with either wild-type or mutant HRE, HIF-1α-expressing constructs, and pRL-TK plasmids under normoxic or hypoxic conditions. Firefly luciferase activity was measured using the Dual Luciferase Reporter Assay System (Promega, Madison, WI, USA) with a luminometer. Renilla luciferase activity served as an internal control. For ChIP assays, cell extracts were cross-linked with 1% formaldehyde. Subsequently, DNA was extracted, sonicated, and immunoprecipitated using antibodies against IgG and HIF-1α, as previously described [15]. The immunoprecipitated DNA was quantified via quantitative real-time PCR using specific primers (Table S1).

2.5 Migration and invasion assays

Migration and invasion assays were performed in a 24-well plate with 12 h and 20 h of incubation as described previously [17]. Cells suspended in serum-free medium were seeded onto Transwell and Matrigel-coated Transwell (Becton Dickinson, Mountain View, CA, USA) for migration (3 × 104 cells) and invasion (5 × 104 cells) assays, respectively. To visualize the migrated and invaded cells, fixed with methanol, and stained with crystal violet (Sigma-Aldrich), the samples were observed under a light microscope.

2.6 Cell viability, cell growth, and colony formation assays

As previously described [18], cell viability was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich) assay. Cells (1 × 104) were seeded onto 24-well plates and cultured for 24, 48, or 72 h. After the respective incubation period, MTT was added to each well at a final concentration of 0.5 µg/ml and incubated for 3 h. Subsequently, cells were lysed with dimethyl sulfoxide, and the absorbance at 550 nm was measured using a microplate reader. To assess cell growth, cells (3 × 105) were seeded onto 6-well plates for indicated times and subsequently counted using the trypan blue exclusion test. For the evaluation of anchorage-independent growth, soft agar colony formation assay was performed as described previously [19]. Cells (5 × 103) suspended in 0.3% agarose gel were plated onto 0.5% agarose gel and incubated for two weeks. Colonies were stained with crystal violet and counted under a light microscope.

2.7 In vivo experiments

All animal experiments were approved by the Institutional Animal Care and Use Committee of the China Medical University. In metastatic mouse models, six-week-old male NOD-SCID mice (obtained from the National Science Council Animal Center, Taipei, Taiwan) were intravenously injected into the tail veins or orthotopically injected with FADU cells (1 × 106). After a 15-week period, the mice were sacrificed, and the presence of lung metastatic nodules was determined. In xenograft models, five-week-old BALB/c nu/nu mice (National Science Council Animal Center) were subcutaneously injected with FADU cells (2 × 106) into the flanks near hind limbs. After 30–35 days of inoculation, the xenografted mice were sacrificed, and tumor volumes were evaluated.

2.8 In-silico analyses

Gene expression RNA-seq and survival data from patients with HNSC and breast cancer were downloaded from The Cancer Genome Atlas (TCGA) database via the Xena platform [20]. Gene Set Enrichment Analysis (GSEA) was conducted using software version 4.3.2, employing the hallmark and KEGG_LEGACY gene sets from the Molecular Signatures Database [21], with phenotype permutation set to 1000 permutations. The ALKBH4 expression in HNSC patients was used as the test variable, and the 5-year survival status of the patients was used as the state variable to calculate the coordinates for the receiver operating characteristic curve analysis using SPSS software. The Youden index [22], selected as the cutoff value, was used to distinguish between high and low ALKBH4 expression. The KM plotter database was used to investigate the correlation between ALKBH4 expression and survival of cancer patients [23].

2.9 Patient samples and immunohistochemical (IHC) staining

Fifteen breast tumor tissues and thirty-one HNSC tissues were collected from Changhua Christian Hospital and China Medical University Hospital. This study was approved by the Institutional Review Board and conducted in compliance with the Helsinki Declaration. Formalin-fixed, paraffin-embedded tissue sections were deparaffinized with three washes of xylene for 5 min each, and then rehydrated. Peroxidase activity was blocked using 3% hydrogen peroxide for 10 min. Sections were incubated with blocking solution (5% FBS in TBST) for 1 h at room temperature. Primary antibodies against HIF-1α (1:150 dilution; Genetex) and ALKBH4 (1:150 dilution; Novus) were applied for overnight incubation at 4 °C. The slides were counterstained with hematoxylin stain solution and detected using the Mouse and Rabbit Specific HRP/DAB IHC Detection Kit - Micro-polymer kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions. Protein expression was represented as H-scores (0–300), calculated by multiplying the percentage of positively stained cells (0–100) by the staining intensity (0 to 3+).

2.10 Statistical analysis

All analyses were carried out using Prism version 8.01 (GraphPad Software Inc., CA, USA). Experiments were conducted in triplicate at a minimum. Error bars indicate the standard deviation (SD) of the data. Statistical comparisons were performed using Student’s t-test, with a p-value < 0.05 considered statistically significant.