Sex Hormone-regulated CMG2 Is Involved in Breast and Prostate Cancer Progression

Abstract

Background/aim: Capillary morphogenesis gene 2 (CMG2) is involved in prostate and breast cancer progression. This study aimed to investigate sex hormone receptor-mediated regulation of CMG2 in breast and prostate cancer, and its implication in disease progression.

Materials and methods: Expression of CMG2, oestrogen receptor (ER) and androgen receptor (AR) was determined in breast and prostate cancer cell lines, respectively, using real-time quantitative PCR (QPCR) and western blot. Association between CMG2 and sex hormone receptors was analysed in a number of transcriptome datasets. Immunochemical staining was performed in tissue microarrays of breast cancer (BR1505D) and prostate cancer (PR8011A). CMG2 expression was determined in 17β-oestradiol treated breast cancer cells and AR over-expressing prostate cancer cells.

Results: CMG2 was found to be inversely correlated with sex hormone receptors in breast and prostate cancer. Lower expression of CMG2 was associated with a poor prognosis in ER (+) breast cancer but not ER (-) tumours. Both ER (+) breast cancer cell lines and AR (+) prostate cancer cell lines presented lower expression of CMG2, which was increased following sex hormone deprivation. Exposure to 17-β-oestradiol and AR over-expression repressed CMG2 expression in breast cancer and prostate cancer cell lines, respectively.

Conclusion: CMG2 is inversely correlated with ER and AR status in breast and prostate cancer, respectively. ER and AR mediate repression of CMG2 expression in corresponding cancerous cells.

Keywords: AR; CMG2; ER; breast cancer; prostate cancer.

Figure 1. Capillary morphogenesis gene 2 (CMG2) is correlated with oestrogen receptor (ER) expression in breast cancer. (A) CMG2 transcript levels in ER (+) and ER (–) breast cancers were analysed in the GEO Dataset GSE20685 using Mann–Whitney test. *p<0.05. (B) Correlation between CMG2 and ESR1 (Erα) in a cohort of breast cancer (GSE20685) was analysed using Spearman test. In TCGA dataset (n=1,044), CMG2 expression levels were evaluated in both ER (+) tumours (C, n=805) and ER (–) tumours (D, n=236). (E) Representative images of the CMG2 immunohistochemistry (IHC) staining taken from ER(+) samples of breast cancer tumours at different T stages. Association with overall survival (OS) was analysed in both ER (+) tumours (F, n=221, cut off value=15) and ER (–) tumours (G, n=284, cut off value=30) using the KMplot (www.kmplot.com). *p<0.05, **p<0.01, ***p<0.001.

Figure 2. Capillary morphogenesis gene 2 (CMG2) expression is regulated by oestrogen in breast cancer cells. (A) CMG2 transcripts were quantitatively analysed in breast cancer cell lines using QPCR. Fold change in gene expression calculated using the 2-ΔΔCT method. (B) CMG2 protein levels in the breast cancer cell lines were analysed using western blot. (C) Correlation between CMG2 and ESR1 (ERα) was analysed in breast cancer cell line data collected from the CCLE dataset using Spearman test. (D) CMG2 expression in MCF-7 cells treated with 17-β-oestradiol (1 nM) was quantified using QPCR. (E) CMG2 expression in Erα-silenced MCF-7 cells with (n=3) (GSE27473, CMG2: 238050_at) was also analysed in comparison with the control (n=3). *p<0.05, **p<0.01, ***p<0.001.

Figure 3. Capillary morphogenesis gene 2 (CMG2) is associated with the progression of androgen receptor (AR)+ prostate cancer. (A) Immunohistochemical staining for CMG2 was performed on a tissue microarray (PR8011a, Biomax) comprising tumours (T), adjacent normal prostatic tissues (AN), and normal prostate tissues (N). (B) Representative images taken from normal, adjacent normal and tumour samples. (C) Expression of ANTXR2 was analysed in a gene expression array dataset (GSE3325, CMG2ANTXR2 gene ID:238050_at, GSE3325). Expression of ANTXR2 was observed in localised primary prostate tumours in comparison with its expression in benign prostate tissues, whilst a further reduction was seen in primary tumours which developed distant metastases. (D) Expression of ANTXR2 in metastases of prostate cancer was analysed in a gene array GEO dataset (GSE6919, CMG2ANTXR2 gene ID: 58617_at, GSE6919). (E) Implication of CMG2 in lymph node metastasis was analysed in tumours of TCGA dataset. The tumours were separated into two groups: AR-high and AR-low groups according to AR expression (cut off value=400.39). (F) Association between CMG2 and Gleason score was also analysed in tumours with different AR expression.

Figure 4. Androgen receptor (AR) mediates a regulation of capillary morphogenesis gene 2 (CMG2) in prostate cancer cells. (A) Correlation between CMG2 and AR as analysed in a gene array dataset (GSE6919, CMG2:51714_at, AR: 52851_at). (B) Expression of CMG2 in AR positive prostate cancer cell lines (LNCAP and VCAP), and AR negative cell lines (PC-3 and DU-145) was determined using QPCR. Fold changes in CMG2 transcripts normalised against GAPDH. AR was over-expressed in both PC-3 and DU-145 cell lines using empty lentiviral vectors or lentiviral vectors carrying the coding sequence of human AR, which is named as AR exp. The expression of AR was determined using both conventional PCR (C) and western blot (D). (E) CMG2 expression was determined in both PC-3 and DU-145 cell lines with AR over-expression using QPCR. (F) CMG2 expression in LNCaP cells following a 24-h deprivation from steroid hormones using charcoal stripped serum was determined using QPCR. *p<0.05, **p<0.01, ***p<0.001.