Estrogen Activation by Steroid Sulfatase Increases Colorectal Cancer Proliferation via GPER

Abstract

Context: Estrogens affect the incidence and progression of colorectal cancer (CRC), although the precise molecular mechanisms remain ill-defined.

Objective: The present study investigated prereceptor estrogen metabolism through steroid sulphatase (STS) and 17β-hydroxysteroid dehydrogenase activity and subsequent nongenomic estrogen signaling in human CRC tissue, in The Cancer Genome Atlas colon adenocarcinoma data set, and in in vitro and in vivo CRC models. We aimed to define and therapeutically target pathways through which estrogens alter CRC proliferation and progression.

Design, setting, patients, and interventions: Human CRC samples with normal tissue-matched controls were collected from postmenopausal female and age-matched male patients. Estrogen metabolism enzymes and nongenomic downstream signaling pathways were determined. CRC cell lines were transfected with STS and cultured for in vitro and in vivo analysis. Estrogen metabolism was determined using an ultra-performance liquid chromatography-tandem mass spectrometry method.

Primary outcome measure: The proliferative effects of estrogen metabolism were evaluated using 5-bromo-2'-deoxyuridine assays and CRC mouse xenograft studies.

Results: Human CRC exhibits dysregulated estrogen metabolism, favoring estradiol synthesis. The activity of STS, the fundamental enzyme that activates conjugated estrogens, is significantly (P < 0.001) elevated in human CRC compared with matched controls. STS overexpression accelerates CRC proliferation in in vitro and in vivo models, with STS inhibition an effective treatment. We defined a G-protein-coupled estrogen receptor (GPER) proproliferative pathway potentially through increased expression of connective tissue growth factor in CRC.

Conclusion: Human CRC favors estradiol synthesis to augment proliferation via GPER stimulation. Further research is required regarding whether estrogen replacement therapy should be used with caution in patients at high risk of developing CRC.

Figure 1.

Estradiol synthesis pathways are upregulated in human CRC. (A) Estrogen metabolism pathways demonstrating the importance of STS and HSD17Bs in estrogen synthesis. (B) STS activity is increased in female (n = 29) and male (n = 31) CRC compared with matched normal colon tissue. *P < 0.05, **P < 0.01, ***P < 0.001 using random effects linear regression modeling. (C) STS activity does not correlate with STS expression (dCT) in CRC or normal colon tissue (n = 62). (D) HSD17B2 (female, n = 19; male, n = 28) expression is downregulated in CRC. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed paired Student t test used). (E and F) HSD17B7 (female, n = 21; male, n = 22) and HSD17B12 (female, n = 21; male, n = 22) expression is upregulated in CRC. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed paired Student t test used). (G and H) Representative blots and relative intensity (arbitrary unit) of HSD17B enzymes in normal and matched cancerous human colon tissue. HSD17B1 (n = 16) protein expression was not present in human CRC, but HSD17B7 was expressed, and HSD17B12 expression was increased (n = 16), with little change in HSD17B2 expression (n = 16). For relative intensity data, a two-tailed Student t test was used. All data presented as mean ± standard deviation.

Figure 2.

Estrogens increase proliferation in CRC cell lines. (A) Expression profile of HSD17B1, HSD17B2, HSD17B7, and HSD17B12 in HCT116, HT29, Caco-2, and Colo205 cells. β-Actin was used as a loading control. One representative blot from three independent experiments. (B and C) E₁ and E₂ increased proliferation rates in a dose-dependent manner in HCT116 and HT-29 cells. Caco-2 cells did not respond to E₁ or E₂ treatment (n = 4 independent experiments). (D and E) HCT116 cells did not readily metabolize E₁, E₂, and E₁S. HT-29 cells metabolized E₁ and E₂ to an unknown metabolite. Caco-2 cells rapidly metabolized E₂ to E₁ (n = 3 independent experiments). All data presented as mean ± standard deviation.

Figure 3.

Overexpression of STS in HCT116 cells increased estrogen-dependent proliferation in vitro and in vivo. (A) HCT116[sts] cells proliferated at a greater rate compared with HCT116[vo] cells. This proliferation was significantly inhibited by STX64 (1 mM). **P < 0.01, ***P < 0.001 (n = 3 independent experiments; one-way analysis of variance, followed by Tukey’s multiple comparison post-test). (B) HCT116[sts] cells had increased proliferation when stimulated with E₂S (100 nM for 72 hours) compared with wild-type HCT116 (HCT116[wt]) and HCT116[vo] cells. This increased proliferation was blocked by STS inhibition using STX64 (1 mM). ***P < 0.001 compared with control; ##P < 0.01, ###P < 0.001 compared with E₂S treatment; ^aP < 0.001 compared with HCT116[vo] (two-tailed Student t test used; n = 4 independent experiments). (C) HCT116[sts] xenografts grew at an increased rate compared with HCT116[vo] xenografts. This increased proliferation was inhibited by STX64 (20 mg/kg thrice weekly, orally). **P < 0.01, ***P < 0.001 (one-way analysis of variance followed by Tukey’s multiple comparison post-test). (D) Five randomly taken tumors imaged after removal. (E) Wet tumor weights at 21 days after HCT116 cell inoculation. HCT116[sts] resulted in an increased tumor burden, which was inhibited by STX64. *P < 0.05, ***P < 0.001 (two-tailed Student t test used). (F) STS activity in HCT116[vo] and HCT116[sts] xenografts at day 21. HCT116[sts] xenograft maintained elevated STS activity compared with HCT116[vo]. STX64 treatment significantly inhibited HCT116[vo] and HCT116[sts] activity. *P < 0.05, **P < 0.01, ***P < 0.001 (n = 5 to 14; two-tailed Student t test used). All data presented as mean ± standard deviation.

Figure 4.

E₂ acts through GPER signaling to increase CRC proliferation. (A) ERα and ERβ were not expressed in HCT116 or HT-29 but were present in Caco2 and Colo205 cells. GPER was expressed in all cell lines tested. β-Actin was used as a loading control. One representative blot from three independent experiments. (B) Schematic of the downstream molecular signaling factors stimulated by GPER action as defined in breast cancer. (C) The GPER agonist G1 increased the proliferation rates in a dose-dependent manner compared with cells grown only in media with sFBS (two-tailed Student t test used; n = 4 independent experiments). (C) The GPER antagonist G15 (1 mM) inhibits the increased proliferation induced by E₂ (100 nM for 72 hours) and G1 (100 nM for 72 hours) in HCT116 and HT-29 cells. **P < 0.01, ***P < 0.001 compared with controls (two-tailed Student t test; n = 4 independent experiments). (D) E₂ (100 nM) and G1 (100 nM) treatment increases CTGF protein expression in HCT116 and HT-29 cells. β-Actin was used as a loading control. One representative blot from three independent experiments. (E) siRNA knockdown of GPER and CTGF in HCT116 cells was achieved for 96 hours after siRNA treatment. (F) siRNA knockdown of GPER and CTGF inhibits E₂ (100 nM) and G1 (100 nM) stimulation of HCT116 proliferation. *P < 0.05, ***P < 0.001 compared with controls (two-way analysis of variance, followed by a Bonferroni post-test; n = 3). (G) G15 (50 mg/kg thrice weekly, intraperitoneally) significantly attenuated HCT116[sts] xenograft tumor growth in female nude mice. **P < 0.01 (two-way analysis of variance; n = 10). (H) Patients with high GPER expression (n = 110) had a significantly worse survival outcome compared with mid to low GPER-expressing (n = 330) CRC tumors, as shown from analysis of the TCGA COAD data set (Kaplan-Meier survival analysis; log-rank method). All data presented as mean ± standard deviation.

Figure 5.

CTGF and GPER expression correlates in human CRC. (A) Immunoblotting of GPER and CTGF expression in normal (N) and cancerous (C) human colon tissue. β-Actin was used as a loading control. One representative blot from three independent experiments. (B) GPER and CTGF expression were increased in human CRC, as measured by the immunoblotting relative intensity to β-actin. *P < 0.05, ***P < 0.001 (two-tailed Student t test used; n = 17). (C) Correlation between GPER and CTGF relative intensity in normal and cancerous human colon tissue (n = 17). (D) Schematic diagram showing proposed pathway through which estrogens act, via GPER, to augment proliferation in CRC.