Cholesterol Synthetase DHCR24 Induced by Insulin Aggravates Cancer Invasion and Progesterone Resistance in Endometrial Carcinoma

Abstract

3β-Hydroxysteroid-Δ24 reductase (DHCR24), the final enzyme of the cholesterol biosynthetic pathway, has been associated with urogenital neoplasms. However, the function of DHCR24 in endometrial cancer (EC) remains largely elusive. Here, we analyzed the expression profile of DHCR24 and the progesterone receptor (PGR) in our tissue microarray of EC (n = 258), the existing EC database in GEO (Gene Expression Omnibus), and TCGA (The Cancer Genome Atlas). We found that DHCR24 was significantly elevated in patients with EC, and that the up-regulation of DHCR24 was associated with advanced clinical stage, histological grading, vascular invasion, lymphatic metastasis, and reduced overall survival. In addition, DHCR24 expression could be induced by insulin though STAT3, which directly binds to the promoter elements of DHCR24, as demonstrated by ChIP-PCR and luciferase assays. Furthermore, genetically silencing DHCR24 inhibited the metastatic ability of endometrial cancer cells and up-regulated PGR expression, which made cells more sensitive to progestin. Taken together, we have demonstrated for the first time the crucial role of the insulin/STAT3/DHCR24/PGR axis in the progression of EC by modulating the metastasis and progesterone response, which could serve as potential therapeutic targets for the treatment of EC with progesterone receptor loss.

Figure 1. DHCR24 is up-regulated in endometrial cancer.

(A) mRNA expression level of DHCR24 in 91 EC tissues and 12 non-tumor tissues in the GEO database. (B) Differential mRNA expression level of DHCR24 in the histological grading of the GEO database. n, number of analyzed samples. (C) mRNA expression level of DHC24 in freshly frozen endometrial cancer tissues. (D) Representative IHC staining of DHCR24 in the tissue microarray (TMA) of endometrial cancer from histological Grade 1 to Grade 3 of EC specimens. Scale bar, 50 μm. (E) Score of the IHC stained in the TMA of EC. Distribution of DHCR24 in the cytoplasm was quantified in EC. Scoring was conducted based upon the percentage of positively stained cells: 0–10% scored 0; 10–35% scored 1; 36–70% scored 2; and more than 70% scored 3. (F) Overall survival Kaplan-Meier estimate of DHCR24 alterations in TCGA (DHCR24 alteration group: Red line, No DHCR24 alteration group: Blue line).

Figure 2. High DHCR24 expression predicts poor prognosis in the two tissue microarrays of EC.

(A) Comparisons of the overall survival (OS) between DHCR24 low- and high-expression groups in EC. (B) Comparisons of the OS between DHCR24 low- and high-expression groups in the histological Grade 2 and Grade 3 cohort. (C) Comparisons of the OS between DHCR24 low- and high-expression groups in the clinical stage II-IV cohort. (D) Comparisons of the OS between DHCR24 the low- and high-expression groups in the vascular invasion cohort.

Figure 3. DHCR24 could be induced by insulin stimulation via STAT3 in EC.

(A) DHCR24 DNA copy number amplification from the genomic-scale sequencing of EC in TCGA. (B) mRNA expression of DHCR24 stimulated by EGF, insulin, IL6, TGF-β and estradiol evaluated by qPCR. (C) The mRNA expression level of DHCR24 and STAT3 under STAT3 siRNA interference. (D) The protein level of DHCR24 was determined in ECC-1 and HEC-1A cells treated with insulin, GSK1904529A and Stattic by immunoblotting (cropped images are shown here, the non-cropped images are shown in Supplementary data 6). (E) A ChIP assay was performed to confirm the potential STAT3 binding site in the DHCR24 promoter region. IgG and input fraction were used as controls. (F) A ChIP assay was performed in two endometrial cancer cell lines, HEC-1A and ECC-1. (G) In total, 10 pairs of primers were constructed according to the promoter of DHCR24. (H and I) A Luciferase reporter assay was performed using ECC-1 and HEC-1A cells after transfecting the wild type plasmids and mutated plasmids (mutation site: red). The data shown are the mean ± SD. (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 4. Silencing of DHCR24 suppresses EC cell invasion and migration.

(A) qRT-PCR analysis of the DHCR24 expression after DHCR24 siRNA interference. (B) Successful DHCR24 silencing was confirmed by immunoblotting. (C) The level of migration was probed in the DHCR24-silenced group and the DHCR24 control group derived from KLE and HEC-1B cells. (D) The level of invasion was assessed in the DHCR24-silenced group and the DHCR24 control group derived from KLE and HEC-1B cells. (E) The level of migration ability of HEC-1B cells treated with insulin, GSK1904529A, and the silencing of DHCR24 was determined using a transwell assay. (F) The invasion ability of HEC-1B cells treated with insulin, GSK1904529A, and silencing of DHCR24 was probed. The data are presented as the mean ± SD of three independent experiments. (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 5. DHCR24 expression level is negatively correlated with PGR expression level.

(A) The correlation between the expression level of DHCR24 and PGR was analyzed in the TCGA dataset of EC (R = −0.395, p = 0.006). (B) The correlation between the expression levels of DHCR24 and PGR was analyzed in the GEO dataset of EC (R = −0.318, p = 0.0021). (C) The correlation between the expression levels of DHCR24 and PGR was analyzed in the EC cell lines (R = −0.775, p < 0.05). (D) mRNA expression level of DHCR24 and PGR under DHCR24 siRNA interference. (E) Representative images of the immunohistochemical staining for DHCR24 and PGR in paraffin continuous sections of Grade I endometrial cancer. (F) Representative images of immunohistochemical staining for DHCR24 and PGR in paraffin continuous sections in Grade III endometrial cancer. Scale bar 50 μm.

Figure 6. Silencing of DHCR24 enhances the sensitivity of endometrial cancer cells to medroxyprogesterone acetate treatment.

(A) The cell viability of HEC-1B cells was determined in the DHCR24-silenced group and the control group treated with different concentration of MPA ranging from 0 to 30 μM. (B) The cell viability of DHCR24-silenced and control KLE cells was measured after treatment with MPA (10 μM). (C) The cell viability of DHCR24-silenced and control HEC1B cells was measured after treatment with MPA (10 μM). (D) The cell viability was determined in HEC-1B cells treated with MPA, insulin, GSK1904529A or Stattic or with silenced DHCR24. (E) Schematic summary of the findings presented in this study on the role of DHCR24 in endometrial cancer. Insulin up-regulated DHCR24 expression via STAT3, directly binding to the DHCR24 promoter region, which modulated cell metastasis and the response to progestin therapy. (**p < 0.01).