Differences in the role of Gper1 in colorectal cancer progression depending on sex

Abstract

To evaluate the role of 17β-oestradiol (E2) in the sex-dependent progression of colorectal cancer (CRC), the present study focused on E2 signalling mediated via the nuclear receptors [oestrogen receptor (ESR)1 and ESR2] and the membrane G protein-coupled oestrogen receptor 1 (Gper1) in males and females diagnosed with CRC. This study also investigated Gper1 signalling in the CRC cell lines DLD1 and LoVo, which differ in the p53 pathway. In cancer tissue, Gper1 becomes by far the most abundant E2 receptor due to an increase in Gper1 and a decrease in ESR2 expression. These changes are more prominent in males than in females. More pronounced differences in Gper1 expression between cancer and adjacent tissues were observed in males in lower stages compared with those in higher stages of disease and females. High expression of Gper1 was associated with worse survival in males without nodal involvement but not in females. The expression of E2 receptors in the CRC cell lines DLD1 and LoVo resembles that of human cancer tissue. Silencing of Gper1 (siGper1) caused an increase in the rate of metabolism in LoVo cells with wild-type tp53. In DLD1 cells with the mutated form of tp53, siGper1 did not exert this effect. High levels of Gper1 were associated with worse survival and could contribute to sex-dependent changes in the CRC prognosis. Tumour suppressor effects of Gper1 were, at least to some extent, dependent on signalling downstream of p53, which was more frequently deficient in males than in females. Overall, this suggests that up-regulation of Gper1 (or administration of a Gper1 agonist) would be more beneficial for patients with wild-type tp53.

Keywords: DLD1; G protein-coupled receptor 30; LoVo; oestradiol; oestrogen receptor 1; oestrogen receptor 2; sex; survival.

Figure 1.

Relative expression of ESR1, ESR2 and Gper1 mRNA in the tumour and corresponding adjacent tissues of patients undergoing surgery for colorectal cancer. (A) Comparison of ESR1, ESR2 and Gper1 mRNA expression in the tumour (white columns) and averaged expression in the corresponding proximal and distal tissues (grey columns). Data are relativised; the averaged expression in samples was set to the same value for all receptors. (B) The abundance of E2 receptors in cancer and adjacent tissues is presented as threshold cycle (Cq-the cycle number of PCR at which the fluorescence reached the threshold in the amplification, the averaged number is shown in the middle of the columns). The threshold was set to 1 in all PCR assays. Data are shown as mean ± standard error of the mean. **P<0.01 and ***P<0.001 cancer tissue compared to adjacent tissue. Columns labelled with different labels are significantly different by ANOVA followed by Tukey's post hoc test at P<0.05. ^#P<0.05 compared with $ and &. ^$P<0.05 compared with # and &; ^&P<0.05 compared with # and $. r.u., relative units; ESR, oestrogen receptor; Gper1, G protein-coupled oestrogen receptor 1; E2, 17β-oestradiol.

Figure 2.

Gper1, ESR1 and ESR2 mRNA expression in cancer and adjacent tissues of (A) males and (B) females. Data are relativised to the same averaged expression in all three datasets (Gper1, ESR1 and ESR2). White columns show expression in cancer and grey columns display mRNA levels in the corresponding proximal and distal tissues. Data are shown as mean ± standard error of the mean. *P<0.05 and ***P<0.001 cancer tissue compared to adjacent tissue. r.u., relative units; ESR, oestrogen receptor; Gper1, G protein-coupled oestrogen receptor 1.

Figure 3.

Comparison of Gper1 mRNA expression in colorectal cancer tissue. Expression of Gper1 compared with adjacent tissues in (A) the whole cohort, (B) males and (C) females according to the TNM classification. White columns show expression in cancer, and grey columns display averaged mRNA levels in the corresponding proximal and distal tissues. Data are shown as mean ± standard error of the mean. *P<0.05 cancer tissue compared to adjacent tissue. T, tumour invasion; N, nodal status; M, distant metastasis; r.u., relative units; Gper1, G protein-coupled oestrogen receptor 1.

Figure 4.

Comparison of Gper1 mRNA expression in colorectal cancer in relation to clinic-pathological characteristics of patients. Gper1 mRNA expression in patients with (A) nodal involvement (B) the presence of distant metastases. White columns show expression in cancer, and grey columns display averaged mRNA levels in the corresponding proximal and distal tissues. Dots are used to show the results for the sub-cohort with nodal involvement, and horizontal hatching indicates the presence of distant metastases. Data are shown as mean ± standard error of the mean. *P<0.05 and **P<0.01 cancer N0/M0 tissue compared to adjacent N0/M0 tissue. r.u., relative units; Gper1, G protein-coupled oestrogen receptor 1.

Figure 5.

Association of overall survival and Gper1 expression evaluated by Kaplan-Meier. Survival curve for the (A) whole cohort without lymph node involvement (N0); (B) whole cohort with lymph node metastases (N1-2); (C) male patients without lymph node involvement; and (D) females without lymph node involvement. The solid line indicates low Gper1 expression (≤ median), and the dotted line indicates high Gper1 expression (> median). Gper1, G protein-coupled oestrogen receptor 1.

Figure 6.

Relative expression of Gper1, ESR1 and ESR2 receptors. mRNA expression of E2 receptors was measured in (A) DLD1 and (B) LoVo colorectal cell lines. Ct=the PCR cycle number at which the fluorescence reached the threshold in the amplification. The threshold was set at 1 in all PCR assays. Columns labelled with different labels are significantly different by ANOVA followed by Tukey's post hoc test at P<0.05. ^#P<0.05 compared with $ and &. ^$P<0.05 compared with # and &; ^&P<0.05 compared with # and $. ESR, oestrogen receptor; Gper1, G protein-coupled oestrogen receptor 1; E2, 17β-oestradiol.

Figure 7.

Effect of Gper1-silencing on the rate of metabolism. Metabolism was measured in (A) DLD1 and (B) LoVo cells by the MTS test. White columns display results from the control group, and grey columns show formazan production in Gper1-silenced cells. Results are presented as mean ± standard error of the mean. *P<0.05 and ***P<0.001 compared with the nc group. r.u., relative units; nc, negative control; si, short interfering; Gper1, G protein-coupled oestrogen receptor 1.

Figure 8.

Effect of Gper1-silencing on the migration. Migration was measured in (A) DLD1 and (B) LoVo cells by the scratch assay. White columns show results from the control group, and grey columns display the rate of wound closure of Gper1-silenced cells. Results are presented as mean ± standard error of the mean. r.u., relative units; Gper1, G protein-coupled oestrogen receptor 1; si, short interfering; nc, negative control.

Figure 9.

Possible mechanism of how Gper1 signalling contributes to sex-dependent differences in CRC. In cancer tissue, the expression of ESR2, which inhibits CRC progression, decreases in the earlier stages of disease in males compared to females (74). E2 signalling is further modulated by an increase in Gper1 expression in cancer tissue in males in the early stages of disease, which is not observed in females. Therefore, in the CRC tumour Gper1-mediated regulation strongly influences how E2 signalling will be interpreted by the cell. Gper1 demonstrates an oncostatic effect in the LoVo cell line carrying wild-type tp53, which is not observed in DLD1 cells with mutated tp53. It was concluded that Gper1 functioning is dependent on the p53 intracellular context. As the mutated form of the gene is more frequent in males compared to females, and the two most important gut receptors, Gper1 and ESR2, show a sex-dependent pattern of expression, we suppose that sex-dependent interpretation of E2 signalling and the availability of a functional p53 pathway contribute cooperatively to the differences in CRC progression observed between sexes. ESR, oestrogen receptor; Gper1, G protein-coupled oestrogen receptor 1; E2, 17β-oestradiol; CRC, colorectal cancer; wt, wild-type.