The lung cancer cell lines (A549 and NCI-H1299), hepatoma cell lines (Hep3B, Huh7, HepG2 and HCCLM3), bladder cancer cell lines (UMUC-3 and T24), colorectal cancer cell lines (T84, LoVo and SW480) and pancreatic cancer cell lines (CFPAC-1 and PANC-1) were purchased from Procell Life Science & Technology Co., Ltd. The tongue squamous cell carcinoma cell lines (HN3, HN4), hepatoma cell line (SNU-449), melanoma cell line (WM35), cervix carcinoma cell line (Hela), epithelial carcinoma cell line (A431), breast cancer cell line (MCF7) and HEK293 cell were maintained by our laboratory. Cell lines were authenticated using short tandem repeat (STR) profiling. MCF7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (FBS) and 1X penicillin/streptomycin and all cells were incubated at 37°C in a humidified 5% CO₂ incubator.

The total RNA from all the aforementioned cancer cell lines was extracted using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) and converted into cDNA by the PrimeScript™ RT reagent kit (Takara Bio, Inc.) according to the manufacturer's protocol. For RT-qPCR, cDNA templates were amplified on the ABI7500 System (Applied Biosystems; Thermo Fisher Scientific, Inc.) using the SYBR Green PCR Kit (Takara Bio, Inc.). The qPCR thermocycling conditions were as follows: 95°C for 2 min, followed by 40 cycles of 94°C for 20 sec, 58°C for 20 sec and 72°C for 30 sec. Relative levels of mRNA expression were calculated using the 2^−ΔΔCq method (19). Primer (10 µM for each gene) sequences for each gene were as follows: HCAR1 forward, 5′-GCCCAGCACTGTTTACCTTTTC-3′ and reverse, 5′-CCCCAAAAGCCCAGTGTCTAC-3′; phosphofructokinase muscle type (PFKM) forward, 5′-GAGTGACTTGTTGAGTGACCTCCAGAAA-3′ and reverse, 5′-CACAATGTTCAGGTAGCTGGACTTCG-3′; PFK liver type (PFKL) forward, 5′-GGCATTTATGTGGGTGCCAAAGTC-3′ and reverse, 5′-CAGTTGGCCTGCTTGATGTTCTCA-3′; hexokinase (HK)2 forward, 5′-GGGCATCTTGAAACAAG-3′ and reverse, 5′-GGTCTCAAGCCCTAAG-3′; pyruvate kinase M2 (PKM2) forward, 5′-GTGGCTCTGGATACAAAGGG-3′ and reverse, 5′-ACTTCTCCATGTAAGCGTTGTC-3′; and β-actin forward, 5′-ACAATGTGGCCGAGGACTTT-3′ and reverse, 5′-TGGGGTGGCTTTTAGGATGG-3′. The β-actin gene was used as an internal control.

Semi-quantitative PCR was performed using Taq PCR mastermix (Tiangen Biotech, Co., Ltd.), The PCR was conducted using the following thermal cycles: Initial denaturation at 94°C for 5 min, followed by 28 cycles of 98°C for 10 sec, 55°C for 15 sec and 72°C for 1 min, and then a final 5-min extension at 72°C. Primer (10 µM for each gene) sequences for semi-quantitative PCR were as follows: HCAR1 forward, 5′-GGAGCATCGTGTTCCTTAC-3′ and reverse, 5′-TTCTTCATCCGAGCCTGT-3′, which amplify a 349-bp fragment; and β-actin forward, 5′-TCTACAATGAGCTGCGTGTG-3′ and reverse, 5′-CAACTAAGTCATAGTCCGCC-3′, which amplify a 878-bp fragment. After amplification, the PCR products were visualized on 1.2% agarose gels containing GelRed (US Everbright, Inc.). The DNA bands were quantitated using ImageJ 1.52a software (National Institutes of Health).

To knock out HCAR1 via CRISPR/Cas9, three sgRNAs were designed using the CRISPR Design Tool (http://crispr.mit.edu/): HCAR1 sgRNA-1, targeting CAGCACGACCCGTTGTACA; sgRNA-2, targeting GGTCGTGCTGCCGCATCGA; and sgRNA-3, targeting CACACAGGACCCGCATCCT. Oligos were annealed at 37°C for 30 min and 95°C for 5 min, and then the temperature was ramped down to 25°C at 5°C/min. The annealed fragments were treated with BpiI endonuclease and incorporated into the pSpCas9(BB)-2A-Puro(pX459) plasmid (Addgene, Inc.). MCF7 cells were seeded in six-well plates and transfected with 1 µg of pX459-HCAR1 plasmid by FuGENE^® HD transfection Reagent (Roche Diagnostics) according to the manufacturer's protocol. Following 3 days after transfection, cells were selected using 2 µg/ml puromycin for 2 weeks. The KO efficiency was validated by genomic sequencing and western blot analysis.

Breast cancer cells were lysed for 30 min on ice in RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% Na deoxycholate, 0.1% SDS, 1 mM PMSF, 1 mM Na₃VO₄, 10 mg/ml leupeptin and 10 mg/ml aprotinin). Protein content was calculated using BCA Protein Assay Kit (Beyotime Institute of Biotechnology). Whole-cell lysates containing 50 µg of proteins were separated on 10% gels using SDS-PAGE and transferred to PVDF transfer membrane (MilliporeSigma) using a semidry transfer system (Bio-Rad Laboratories, Inc.). After blocking in a 5% BSA (Biosharp Life Sciences) solution in 1X TBST for 1 h at room temperature, the membrane was probed with anti-HCAR1 (1:1,000; cat. no. SAB1300090; Sigma-Aldrich; Merck KGaA) or β-actin (1:1,000; ca. no. 4967; Cell Signalling Technology, Inc.) antibody for 1 h at room temperature and subsequently with HRP-conjugated secondary antibodies (1:2,000; cat. nos. 7076/7074; Cell Signalling Technology, Inc.) for 1 h at 37°C. Immunoreactive bands were detected using an enhanced chemiluminescent (ECL) reagent (Thermo Fisher Scientific, Inc.) by using Azure Biosystem C600 (Azure Biosystems, Inc.). Western blots were quantitated using ImageJ 1.52a software (National Institutes of Health).

Cell viability was assessed using a CCK-8 assay (Dojindo Laboratories, Inc.). Briefly, cells were seeded at 1,000 cells/well in 96-well plates and were incubated for different time periods (24, 48, 72 and 96 h) at 37°C. CCK-8 (10 µl) solution was added to each well and then incubation followed for 2 h. The absorbance value (OD) of each well was measured at 450 nm with a microplate reader. Each sample was assayed in four replicates. Three independent experiments were performed.

The EdU incorporation assay was carried out by a Click-iT^® EdU Alexa Fluor^® 555 Imaging Kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Briefly, MCF7 cells were seeded into 24-well plates at 1×10⁴ cells/well and incubated at 37°C overnight. After treatment with 20 mM lactate or DMEM for 24 h, the cells were treated with EdU (10 mmol/l) for 1 h. Then, the cells were fixed at room temperature for 15 min, permeabilized and incubated with the Click-iT reaction cocktail (Invitrogen; Thermo Fisher Scientific, Inc.), followed by Hoechst 33342 (Invitrogen; Thermo Fisher Scientific, Inc.) staining, and observed using a fluorescence microscope (Nikon Corporation). The number of EdU-positive cells and Hoechst 33342-positive cells were counted using ImageJ 1.52a software (National Institutes of Health).

The colony formation ability of HCAR1-KO and control MCF7 cells was estimated by a colony formation assay. Briefly, cells pretreated with lactate (50 mM, with a stronger effect) for 24 h were seeded in a six-well plate at a density of 300 cells/well, followed by ten days of culture. Then, the clones (a colony was defined as >50 cells) were stained with 0.1% crystal violet (Solai Bao Technology Co., Ltd.) for 30 min at room temperature and counted using ImageJ 1.52a software. The data shown represent the average of three independent experiments.

Cell cycle distribution was assessed with a Cell Cycle Analysis kit (BD Biosciences) by flow cytometry. Cells were harvested and washed with PBS containing 1% FBS. Then, the cells were fixed with 70% ethanol at 4°C overnight, washed twice with PBS and incubated with propidium iodide (PI)/RNase A staining solution (5 µg/ml PI, 250 µg/ml RNase A in PBS) for 15 min at 37°C in the dark. The level of PI incorporation was analyzed by FACScan (BD FACS Canto II). The percentage of cells in the respective cell cycle phase was determined using Modfit LT version 3.2 software (Verity Software House, Inc.).

Transwell chambers (MilliporeSigma) were used to investigate cell migration ability. A total of 1×10⁵ cells were washed with serum-free medium and treated with 100 ng/ml pertussis toxin (PTX) for 6 h at 37°C. Cells were plated in the upper chambers (8.0 µm). Medium containing 10% FBS with 2 or 20 mM lactate was added to the lower chambers to serve as a chemoattractant. After 24 h of incubation, migratory cells in the lower chambers were fixed, stained with 0.1% crystal violet solution (Solai Bao Technology Co., Ltd.) for 30 min and quantified using light microscopy. The cell numbers were counted in 5 different random fields (magnification, ×200).

MCF7 cells were seeded at a density of 2.0×10⁵/well in six-well plates. The cells were then collected and the enzyme activity of PFK, HK and PK were determined using test kits from Nanjing Jiancheng Bioengineering Institute (catalog nos. A129-1-1, A077-3-1 and A076-1-1) according to the manufacturer's instructions.

The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of cells were evaluated using a Seahorse XF96 Extracellular Flux Analyzer (Seahorse Bioscience). Briefly, MCF7 cells were seeded at a density of 1×10⁴ cells/well into 96-well Seahorse microplates for 24 h, and the calibration plate was equilibrated overnight in a non-CO₂ incubator. Cells were washed twice with assay running media and equilibrated in a non-CO₂ incubator before starting the test. Once the probe calibration was completed, the calibration plate was replaced with the cell plate to simultaneously measure the OCR and ECAR of the cells. After injection of oligomycin (1 µM), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (1 µM), rotenone (1 µM) and antimycin A (1 µM), OCR and the corresponding ECAR were determined. Once completed, the protein concentration was quantified (BCA Protein Assay Kit; Thermo Fisher Scientific, Inc.) to normalize the OCR and ECAR values.

All data were analyzed using SPSS pack 26.0 software (IBM Corp,), and the graphs were constructed by GraphPad Prism 5.0 software (GraphPad Software, Inc.). Data are presented as the mean ± SD and represent at least three independent experiments. Unpaired Student's t-tests, or one-way ANOVA with Bonferroni's post hoc test was used as indicated. P<0.05 was considered to indicate a statistically significant difference.

To determine whether HCAR1 was expressed in cancer cells of solid tumours, whose interior are hypoxic and shows more lactate accumulation (20,21), HCAR1 mRNA levels were analyzed in various cancer cell lines. It was found that HCAR1 was expressed in tongue, lung, breast, bladder, pancreatic, hepatocellular, colorectal, cervical and epidermal carcinoma cells (Fig. 1A). Due to high HCAR1 expression, breast cancer cells were selected for further study. Compared with all kinds of breast cancer cell lines, a higher level of HCAR1 mRNA and protein expression was observed in MCF-7 cells (Fig. 1B and C). These results demonstrated that HCAR1 was expressed in numerous cancer cell types and in almost all human breast cancer cells.

To elucidate the potential effect of HCAR1 on cell proliferation, HCAR1-KO MCF7 cells were generated by the CRISPR/Cas9 system. A total of 3 lenti-CRISPR/Cas9-KO constructs containing nonoverlapping sgRNAs (‘sgRNA1/2/3’) were utilized to establish stable HCAR1-KO MCF7 cells (Figs. 2C, S1A and B). The survival of HCAR1-KO MCF7 cells treated with lactate was analyzed by CCK-8 assay (Fig. 2A). HCAR1 depletion potently inhibited the viability of MCF7 cells, and lactate incubation significantly promoted the proliferation of wild-type (WT) but not HCAR1-KO cells. Consistent with these findings, colony formation and EdU assay results further demonstrated that HCAR1 KO inhibited MCF7 cell proliferation, and lactate treatment did not have a proliferation-promoting effect compared with WT MCF7 cells (Fig. 2B and D). As proliferation-suppression phenotypes were observed after depletion of HCAR1, cell cycle distribution was further detected by PI staining and flow cytometry. Consistently, a significant increase in the G1 phase and a decrease in the G2 phase were observed in MCF7 HCAR1-KO cells (Fig. 2E). These results indicated that HCAR1 promoted MCF7 cell proliferation.

The effects of HCAR1 on the migratory ability of MCF7 cells were assessed by a Transwell migration assay. The depletion of HCAR1 in MCF7 cells resulted in a significant reduction in cell migration (Fig. 3A). However, the inhibition of cell migration was attenuated after re-overexpression of HCAR1 (Figs. 3B and S2). In addition, lactate promoted WT MCF7 cell migration in a lactate concentration-dependent manner, which was inhibited after Gi inhibitor-PTX treatment (Fig. 3C). These data indicated that HCAR1 played a vital role in breast cancer cell migration and that the Gi protein may participate in the regulation of MCF7 cell migration.

The speed and direction of the metabolic reaction are associated with rate-limiting enzymes such as PFK, HK and PK (22,23). Therefore, it was investigated whether HCAR1 regulated the expression of those key rate-limiting enzymes in glucose catabolism by RT-qPCR. As revealed in Fig. 4A-D, there was a significant decrease in the mRNA expression of PFKL and HK2 in MCF7 HCAR1-KO cells, whereas a significant inhibition of the enzyme activity of PFK and HK was also observed in HCAR1-KO cells.

The glycolysis metabolism results suggested that HCAR1 could regulate the expression and activity of glycolytic enzymes. To evaluate whether KO of HCAR1 reversed metabolic reprogramming consistent with the Warburg effect, cell energy phenotype assays were conducted using the XFp seahorse bioanalyzer system. This assay delineated the metabolic phenotypes of MCF7 cells under baseline, KO and overexpression conditions. These results indicated that HCAR1 KO reduced the ECAR of MCF7 cells and augmented the OCR compared with control cells, which shifted their energy derivation towards OXPHOS (Fig. 4G and H). Moreover, HCAR1 overexpressing cells had an increased ECAR and a reduced OCR compared with WT cells (Fig. 4E and F). These results indicated that HCAR1 could metabolically control breast cancer by favoring glycolysis over OXPHOS.