Tissue specimens from 136 cases of breast cancer collected from Sapporo Medical University Hospital (Sapporo, Japan) during surgical resection from 2011–2014 were used in the present study. Data were also collected from the pathology file of Sapporo Medical University Hospital. The mean age of the patients was 59.3 years (range, 26–92 years). Histological type was based on the World Health Organization (WHO) classification of tumors of the breast (5th edition) (16). For intrinsic subtype classification, surrogate molecular breast cancer classification based on immunohistochemical assessment of the estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and Ki-67 biomarkers was used according to the WHO classification of tumors (5th edition) (16). All of the 136 cases were staged according to the Union for International Cancer Control stage classification (7th edition) in the WHO classification of tumors (5th edition) (Table I) (16). In the staging process of breast tumors, we categorized tumors using parameters such as pT factor, which is defined as pathological status of primary tumor, and pN factor, which is defined as pathological status of lymph node involvement (16). The present study was approved by the Ethics Committee (approval no. 4-1-44) and Institutional Review Board (study no. 312-230) of Sapporo Medical University (Sapporo, Japan). The Ethics Committee waived the requirement to obtain written informed consent from the patients for the use of human tissues owing to the retrospective nature of the study. The research was performed in accordance with the Declaration of Helsinki. The researchers involved in this study had no access to information that could identify individual participants during or after data collection.

Tissue sections were fixed with 10%-buffered formalin overnight at room temperature. Fixed tissues were embedded in paraffin and embedded sections were cut at 5 µm thickness. Tissue sections were then deparaffinized in xylene, rehydrated using a decreasing ethanol series and incubated in 3% H₂O₂ for 10 min to block endogenous peroxidase activity. After antigen retrieval by microwave heating (95°C for 30 min) in 10 mmol/l Tris and 1 mmol/l EDTA buffer, the sections were incubated overnight at 4°C with primary monoclonal antibodies against CYP24A1 (1:100; cat. no. sc-365700; Santa Cruz Biotechnology, Inc.). The sections were then incubated with EnVision (Dako; Agilent Technologies, Inc.) for 30 min at room temperature, and 3,3′-diaminobenzidine tetrahydrochloride (Dako; Agilent Technologies, Inc.) was added as the chromogen. Hematoxylin was used for counterstaining at room temperature for 3 min. Analysis of immunohistochemical staining positivity was performed using a light microscope, based on the staining intensity and the percentage of positive cells. The intensity scores of staining were set as follows: 3+, strong; 2+, moderate; 1+, weak; and 0, negative. The observers were blinded to the clinical data during the evaluation. Consensus was reached by discussion of discordant cases.

The ER positive, breast cancer MCF7 cell line, was purchased from a local distributor (Summit Pharmaceuticals International Corporation) of the American Type Culture Collection. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM, MilliporeSigma) supplemented with 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.) and 5% streptomycin (MilliporeSigma). The cells were transfected with different types of CYP24A1-specific small-hairpin RNA (shRNA)-expressing lentivirus plasmids according to manufacturer's instruction provided (MISSION^® shRNA Plasmid DNA; MilliporeSigma). Transfection was performed with 5 µg shRNA plasmid, using FuGENE6 (Roche Diagnostics) to generate stable transfectants. Cells were incubated with plasmid for 48 h at 37°C in a humidified 5% CO₂ atmosphere. The shRNAs used were as follows: CYP24A1 shRNA #1296 (shRNA clone ID: NM_ 000782.2-1296s1c1, MilliporeSigma) with the plasmid sequence, 5′-CCGGGCAGATTTCCTTTGTGACATTCTCGAGAATGTCACAAAGGAAATCTGCTTTTTG-3′ and CYP24A1 shRNA #1016 (shRNA clone ID: NM_000782.2-1016s1c1, MilliporeSigma) with the plasmid sequence, 5′-CCGGCGAACTGAACAAATGGTCGTTCTCGAGAACGACCATTTGTTCAGTTCGTTTTTG-3′. Tran-sfected clones were selected using 1.5 µg/ml puromycin (MilliporeSigma). Drug-resistant clones were selected after ≥14 days of selection and screened for CYP24A1 expression to measure their CYP24A1 RNA expressions using reverse transcription (RT)-PCR analysis. Following screening, the MCF7 cell transfectants, CYP24A1 shRNA #1 and CYP24A1 shRNA #7 were used in subsequent experiments. We have previously reported that the process of transfection with CYP24A1 scramble shRNA did not affect the cell phenotype (15). Therefore, wild-type MCF7 cells were used as the control in the present study.

Total RNA of wild-type MCF7 cells and their transfectants was isolated using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and subsequent RT-PCR was performed using the Superscript II Reverse Transcriptase kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to manufacturer's protocols. Samples were incubated at 42°C for 50 min followed by incubation at 70°C for 15 min. The complementary DNA was then mixed with the primers as follows: CYP24A1 forward (F), 5′-GCAGCCTAGTGCAGATTTCC-3′ and reverse (R), 5′-ACCAGGGTGCCTGAGTGTAG-3′; and GAPDH F, 5′-GTCTCCTCTGACTTCAACAGCG-3′ and R, 5′-ACCACCCTGTTGCTGTAGCCAA-3′, as well as 0.5 U of Taq DNA polymerase (Takara Bio, Inc.) to amplify the genes of interest. The thermocycling conditions used were as follows: 40 cycles at 96°C for 30 sec, 30 sec at 55°C, and 1 min at 72°C, followed by a final elongation stage at 72°C for 7 min. RNA expression was examined by loading on 1.5% agarose gels and visualized by ethidium bromide staining. As a loading control, 50 ng of 200 bp DNA ladder (Takara Bio, Inc.) was used. Finally, RNA expression levels were semi-quantified using ImageJ software (version 1.52; National Institutes of Health) and normalization to GAPDH expression.

Cells were seeded in 35 mm dishes (10,000 cells/dish) containing 15 mm glass coverslips (AGC Techno Glass Co., Ltd.) and incubated with DMEM containing 10% FBS (Invitrogen; Thermo Fisher Scientific, Inc.). Glass coverslips were pre-coated with 1:1 rat tail collagen overnight at room temperature (Invitrogen; Thermo Fisher Scientific, Inc). The cells on the coverslips were fixed at 20°C for 10 min with a fixing solution (acetone:ethanol, 1:1). The cells were incubated with a primary monoclonal anti-CYP24A1 antibody (1:100, cat. no. sc-365700, Santa Cruz Biotechnology, Inc.) at 4°C overnight, and then treated with Alexa Fluor 488 (green)-conjugated anti-rabbit IgG (1:200, cat. no. A-11008, Invitrogen; Thermo Fisher Scientific, Inc.) for 1 h at room temperature. The nuclei in the cells were counterstained using 4′,6-diamidino-2-phenylindole at room temperature for 5 min (MilliporeSigma). The samples were imaged using an epifluorescence microscope (Olympus Corporation).

To evaluate cell viability, cells were seeded in 12-well dishes (40,000 cells/well) and the cells were counted by manual cell counting using trypan blue dye exclusion test (cells were stained with trypan blue at room temperature for 1 min) in a time and dose-dependent manner (dependent on the treatment conditions) using a light microscope (Olympus). To assess cell viability under different levels of cell stress, cells were treated with H₂O₂ (0, 25, 50 or 75 µM) for 4, 8 and 12 h to induce oxidative stress at 37°C in a humidified, 5% CO₂ atmosphere. For the assessment of cell proliferation with and without treatment with vitamin D (1 µM, MilliporeSigma) and/or ketoconazole (2 µM, MilliporeSigma), cells were seeded in 12-well dishes (1,000 cells/well). The cells were treated with 1 µM vitamin D and 2 µM ketoconazole and incubated for 48 h at 37°C in a humidified, 5% CO₂ atmosphere.

For the assessment of drug sensitivity, cells were seeded in 96-well plates (5,000 cells/well) and treated with cisplatin (0–100 nM, Adipogen AG) and gefitinib (0–100 nM; Cayman Chemical Company). The viability of cells treated with cisplatin was assessed every 24 h until 96 h of incubation and the viability of cells treated with gefitinib was assessed after 48 h. Cell viability was analyzed using a Cell Counting Kit-8 assay kit (Dojindo Laboratories, Inc.) according to the manufacturer's protocols. Absorbance at a wavelength of 450 nm was quantified using a Varioskan™ LUX microplate reader (Thermo Fisher Scientific, Inc.).

Cells were treated with H₂O₂ (0, 100 or 750 µM) for 24 h at 37°C in a humidified, 5% CO₂ atmosphere to induce oxidative stress. Cells were harvested from culture dishes using a cell lifter and then collected by centrifugation at 300 × g for 3 min at room temperature. The collected cells were solidified using 10% agarose gel and fixed in 10% buffered formalin overnight at 4°C. These tissues were paraffin-embedded and the tissue sections were cut at 5 µm thickness. Immunostaining was performed using primary antibodies against cleaved caspase-3 (1:50, cat. no. 9664; Cell Signaling Technology, Inc.) and Ki-67 (MIB-1 clone; 1:200, cat. no. AM297-5M, BioGenex Laboratories) at room temperature for 1 h. The sections were then incubated with EnVision (1:1, cat. no. K400311, Dako; Agilent Technologies, Inc.) at room temperature for 30 min. After washing with PBS, 3,3′-diaminobenzidine tetrahydrochloride (1:1, cat. no. GE001, Dako; Agilent Technologies, Inc.) was added as the chromogen at room temperature for 5 min.

Cells were seeded in 12-well plates (2,500 cells/well). After incubation of cells for 7 days at 37°C in a humidified 5% CO₂ atmosphere, the cells were fixed using 10% buffered formalin for 15 min at room temperature and stained using 0.04% crystal violet for 15 min at room temperature. Cell clusters >50 µm in diameter were defined as positive; colonies were counted using phase-contrast microscopy (Olympus Corporation) under low magnification (×100) and were quantified using ImageJ software (version 1.52; National Institutes of Health).

At least 3 independent experiments were performed for each analysis and all data were presented as mean ± standard deviations. All data from each experiment were analyzed with either unpaired Student's t-test, Fisher's exact test or the Kruskal-Wallis test to determine significance. Bonferroni's post-hoc test was used where appropriate. Survival curves were constructed, and the Kaplan-Meier method and log-rank test were used to calculate the survival rate. R (version 4.0.3; RStudio, Inc.) was used for all statistical analyses. P<0.05 was considered to indicate a statistically significant difference.

Previous studies reported that CYP24A1 is highly expressed in different types of cancer (13–15). In the present study, the correlations between CYP24A1 expression and the clinicopathological parameters of breast cancer were evaluated using immunohistochemistry (Table I and Fig. 1A-C). CYP24A1 was strongly expressed but its expression area was limited in normal ductal and acinar cells (data not shown). In non-invasive breast carcinoma, samples were positive for CYP24A1 expression 58.6% (17/29) of the cases. Consistent with previous reports, the CYP24A1-positive rates were 83.5% (86/103) and 100% (3/3) in invasive ductal carcinoma and invasive lobular carcinoma, respectively (P=0.0067, Fisher's exact test). No significant association of CYP24A1 expression with primary tumor status and lymph node involvement was demonstrated; however, tumor stage was significantly positively associated with a high expression level of CYP24A1 (P=0.0294, Kruskal-Wallis test).

Based on immunohistochemical assessment of the biomarkers ER, PR, HER2 and Ki-67, breast tumors can be classified into four major immunohistochemically surrogate intrinsic subtypes as follows: Luminal A (ER+, PR+/-, HER2-), Luminal B (ER+, PR+/-, HER2-, higher Ki-67 expression), HER2 (ER-, PR-, HER2+) and basal (ER-, PR-, HER2-) (16). Although some are overlapping, the prognosis of breast cancer patients has been reported to become worse in the order of luminal A, luminal B, HER2-overespressed type, and basal subtype (15–19). In the present study, intrinsic surrogate subtype was associated with CYP24A1 expression, with the expression of CYP24A1 being significantly higher in subtypes with poor prognosis. Indeed, a significant increase in the expression of CYP24A1 was noted in hormone receptor negative cancer (P=0.0047, Kruskal-Wallis test).

The cases were divided into two groups based on the expression of CYP24A1 assessed by staining intensity and area. Specimens containing >50% area with staining intensity 3+ were defined as positive, and specimens containing ≤50% area with staining intensity 3+ were defined as negative. Kaplan-Meier survival curves demonstrated that the overall survival rate in the CYP24A1-positive group was markedly lower than that in the CYP24A negative group when compared in whole specimens (Fig. 1D). However, a significant association between the CYP24A1 positivity and overall survival rate was demonstrated in invasive breast carcinoma (P=0.0266; Fig. 1E). These results were consistent with results of previous studies which suggested a possible oncogenic effect of CYP24A1 in the growth of a breast neoplasm (13–15).

For the establishment of MCF7 cells with suppressed CYP24A1 expression, cells were transfected with shRNAs against CYP24A1 with two different sequences and the cell lines were denoted as CYP24A1 shRNA #1 and CYP24A1 shRNA #7. An immunofluorescent assay demonstrated that the expression of CYP24A1 protein was markedly suppressed in both cell lines (Fig. 2A). RT-PCR analysis demonstrated that CYP24A1 RNA expression levels were markedly reduced in CYP24A1 shRNA #7 (~75%) and CYP24A1 shRNA #1 cells (~50%) (Fig. 2B).

To evaluate the effect of CYP24A1 knockdown on cell viability, MCF7 cells were cultured for 12 h with and without oxidative stress, induced using H₂O₂ (Fig. 3A). In the absence of H₂O₂, no difference in cell viability was demonstrated among all cell groups. The viability of CYP24A1 shRNA #7 cells cultured with H₂O₂ significantly decreased in a dose and time-dependent manner (Fig. 3B and C). However, there was no significant difference in the viability of CYP24A1 shRNA #1 cells cultured with and without H₂O₂.

Cells were treated with vitamin D and ketoconazole, a broad-spectrum inhibitor of CYP24A1, and a manual cell count was performed after 48 h. Although ER-positive cells such as MCF7 cells are known to express higher levels of VDR than the levels in ER-negative cells (1,16), vitamin D only demonstrated a marked decreased in the viability of CYP24A1 shRNA #7 cells. There was no marked difference in cell viability when ketoconazole was added (Fig. 3D).

Cell blocks from cultured cells were established to assess cell death sensitivity under cell stress conditions and the number of apoptotic bodies were manually counted (Fig. 4A). Apoptosis was markedly increased in CYP24A1 knockdown cells. The number of apoptotic cells markedly increased in cells with suppression of CYP24A1 expression when a moderate level of oxidative stress (100 µM H₂O₂) was added. In controls, the number of apoptotic cells only increased with a higher level of oxidative stress (750 µM H₂O₂) (Fig. 4B). These results suggested that cells with suppression of CYP24A1 expression had a higher cell death sensitivity to a cell stressor.

Immunohistochemistry was performed using an antibody against cleaved caspase-3 to evaluate the effects of altered CYP24A1 expression on apoptosis (Fig. 4C). The proportion of cleaved caspase-3-positive cells was significantly increased in CYP24A1 knockdown cells treated with H₂O₂ (Fig. 4D).

To evaluate the effect of CYP24A1 knockdown on two-dimensional tumorigenicity with and without vitamin D and ketoconazole treatment, a colony formation assay was performed (Fig. 5). Compared with the colony-forming ability of the control cells without treatment, this ability was markedly suppressed in both CYP24A1 shRNA #1 and shRNA #7 cells when untreated. Furthermore, colony formation efficacy was suppressed more in CYP24A1 shRNA #7 cells compared with that by CYP24A1 shRNA #1 cells. In the presence of vitamin D, the area of the colonies was markedly decreased in all cell groups. The results suggested that cellular vitamin D status effected colony formation efficacy. However, ketoconazole did not affect colony formation compared with the untreated groups.

To evaluate cell sensitivity to anticancer drugs with different pharmacological mechanisms (cisplatin and gefitinib), cells were cultured with each drug and cell viability was analyzed (Fig. 6). Reduced expression of CYP24A1 significantly enhanced cell death sensitivity to both cisplatin and gefitinib, compared with the control.