Diminished expression of CRHR2 in human colon cancer promotes tumor growth and EMT via persistent IL-6/Stat3 signaling

Abstract

Background & aims: Chronic inflammation promotes development and progression of colorectal cancer (CRC). We explored the distribution of Corticotropin-Releasing-Hormone (CRH)-family of receptors and ligands in CRC and their contribution in tumor growth and oncogenic EMT.

Methods: mRNA expression of CRH-family members was analyzed in CRC (N=56) and control (N=46) samples, 7 CRC cell lines and normal NCM460 cells. Immunohistochemical detection of CRHR2 was performed in 20 CRC and 5 normal tissues. Cell proliferation, migration and invasion were compared between Urocortin-2 (Ucn2)-stimulated parental and CRHR2-overexpressing (CRHR2+) cells in absence or presence of IL-6. CRHR2/Ucn2-targeted effects on tumor growth and EMT were validated in SW620-xenograft mouse models.

Results: CRC tissues and cell lines showed decreased mRNA and protein CRHR2 expression compared to controls and NCM460, respectively. The opposite trend was shown for Ucn2. CRHR2/Ucn2 signaling inhibited cell proliferation, migration, invasion and colony formation in CRC-CRHR2+ cells. In vivo, SW620-CRHR2+ xenografts showed decreased growth, reduced expression of EMT-inducers and elevated levels of EMT-suppressors. IL-1b, IL-6 and IL-6R mRNAs where diminished in CRC-CRHR2+ cells, while CRHR2/Ucn2 signaling inhibited IL-6-mediated Stat3 activation, invasion, migration and expression of downstream targets acting as cell cycle- and EMT-inducers. Expression of cell cycle- and EMT-suppressors was augmented in IL-6/Ucn2-stimulated CRHR2+ cells. In patients, CRHR2 mRNA expression was inversely correlated with IL-6R and vimentin levels and metastasis occurrence, while positively associated with E-cadherin expression and overall survival.

Conclusions: CRHR2 downregulation in CRC supports tumor expansion and spread through maintaining persistent inflammation and constitutive Stat3 activation. CRHR2^low CRC phenotypes are associated with higher risk for distant metastases and poor clinical outcomes.

Keywords: colorectal cancer; inflammation; metastasis; neuropeptides.

Figure 1

Corticotropin-releasing hormone receptor 2 (CRHR2) and urocortin-2 (Ucn2) are differentially expressed in colorectal cancer (CRC) patients. (A) Statistically significant inhibition (***P < .001) of CRHR2 mRNA levels in CRC tissues (n = 56) compared with normal controls (n = 46). (B) Statistically significant overexpression (*P = .016) of Ucn2 mRNA in CRC tissues (n = 56) compared with normal controls (n = 46). (C) CRHR2 protein down-regulation in CRC tissues (n = 20) over normal (n = 5) (P = .0001). (D) Representative CRHR2 staining in CRC and normal tissue arrays. Original magnification ×20, scale bar: 50 μm (scale bar proportional to the original microscope-derived image).

Figure 2

Corticotropin-releasing hormone receptor 2 (CRHR2) and urocortin-2 (Ucn2) are differentially expressed in colorectal cancer (CRC) cell lines. (A) Statistically significant inhibition of CRHR2 mRNA expression in seven CRC cell lines (SW480, SW620, SW403, HT29, DLD1, HCT116, and CaCo2) compared with an immortalized colonic epithelial cell line NCM460. *P < .05; **P < .01. (B) Ucn2 is statistically significantly overexpressed in the CRC cell lines compared with NCM460. *P < .05. (C) Representative Western blot showing CRHR2 down-regulation at the protein level in CRC cell lines over NCM460. Densitometric analysis corresponds to the presented Western blot. Lanes of bands shown have been cropped from the original whole gel image. (D) Fold change of Ucn2 protein expression in CRC cell culture supernatants compared with NCM460. **P < .01; ***P < .001. (E) CRC cells with profiles of CRHR2 down-regulation and Ucn2 up-regulation versus NCM460 were lentivirally transduced to overexpress CRHR2 (CRHR2⁺ cells). Induced expression was validated by quantitative reverse-transcription polymerase chain reaction. EV stands for parental cells transduced with an empty (CRHR2⁻) virus. (F) Statistically significant elevation of cAMP production in CRHR2⁺ versus EV cells as response to increasing Ucn2 concentrations. **P < .01. (G) CRHR2 induction lowers Ucn2 baseline mRNA levels in SW480 and SW620 cells. *P < .05.

Figure 3

Corticotropin-releasing hormone receptor 2 (CRHR2) induction in colorectal cancer (CRC) cells inhibits tumor growth and oncogenic epithelial-to-mesenchymal transition (EMT) progression. (A) CRHR2 induction in SW480 and SW620 inhibits specifically baseline and urocortin-2 (Ucn2)-mediated cell proliferation. Cells were treated with 0.1 μM Ucn2 ± 1 μM astressin-2B (Ast2B) for 72 hours. (B) NCM460-CRHR2-overexpressing cells have increased and CRHR2-specific baseline and Ucn2-mediated proliferation rates compared with parental NCM460 cells. *P < .05; **P < .01. (C) SW480-CRHR2⁺ and SW620-CRHR2⁺ cells are less efficient in forming colonies than corresponding parental EV (empty virus) cells. Cells were treated with 0.1 μM Ucn2 every 2 days for 15 days. (D) SW620- and HCT116-overexpressing CRHR2 showed reduced baseline and Ucn2-mediated migration and (E) invasion potential over parental cells. Both responses were reversed by Ast2B, thus suggesting CRHR2 specificity. In both assays, cells in upper chambers were treated with 0.1 μM Ucn2 ± 1 μM Ast2B for 48 hours. *P ≤ .05; **P ≤ .01. (F) CRHR2 overexpression enhances specifically the migratory properties of NCM460 cells. *P < .05; **P < .01. (G) CRHR2 induction reduces Snail/transforming growth factor β1 (TGFβ1)-mediated migration of SW480 cells. SW620-EV and SW620-CRHR2⁺ cells were transfected with a plasmid vector expressing SnailS6A and incubated with or without 15 ng/mL TGFβ1 and/or 0.1 μM. Migration was assessed 48 hours after transfection. *P ≤ .05; **P ≤ .01.

Figure 4

Corticotropin-releasing hormone receptor 2 (CRHR2)/urocortin-2 (Ucn2) signaling inhibits specifically colorectal cancer (CRC) growth and expression of epithelial-to-mesenchymal transition (EMT) markers in vivo. (A) Reduced tumor volume in mice bearing SW620-CRHR2⁺ xenografts with or without intratumoral Ucn2 administration. Black arrow indicates treatment initiation. Statistically significant differences in tumor volume were observed at weeks 4, 5, and 6 between the following groups: EV versus CRHR2⁺ (P < .003), EV/Ucn2 versus CRHR2⁺/Ucn2 (P < .001), and CRHR2⁺/Ucn2 versus CRHR2⁺/Ucn2/Ast2B (P < .004). (B) Increased necrosis in resected SW620-CRHR2⁺ xenograft specimens versus EV (empty virus) control, as assessed by H&E staining. Scale bar: 2 mm. *P < .05. (C) Increased tumor apoptosis in SW620-CRHR2⁺ specimens, as assessed by terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling assay. Scale bar: 50 μm. *P < .05. (D) Low proliferating cell nuclear antigen (PCNA) expression in SW620-CRHR2⁺ specimens indicates reduced tumor proliferation. Scale bar: 50 μm. Black arrow indicates the specific PCNA staining. (E) Induced expression of the metastasis-suppressor Raf-1 kinase inhibitory protein (RKIP) and inhibition of the EMT-inducers vimentin, N-cadherin, and Snail as well as the angiogenesis promoting VEGF in SW620-CRHR2⁺ specimens derived from Ucn2-treated mice. Scale bars: 50 μm (scale bars are proportional to the original microscope-derived images).

Figure 5

Corticotropin-releasing hormone receptor 2 (CRHR2) suppresses inflammatory responses and interleukin-6 (IL-6)-mediated signal transducer and activator of transcription 3 (Stat3) activation in colorectal cancer (CRC). (A) Decreased IL-6 and IL-1b mRNAs in CRHR2⁺ versus EV (empty virus) CRC cells. *P < .05; **P < .01. (B) Inhibition of IL-6R mRNA expression in CRHR2⁺ cells compared with EV. **P < .01. (C) Urocortin-2 (Ucn2) inhibition by small interfering RNA diminishes IL-6, IL-1b, and IL-8 mRNA expression in SW480 cells (*P < .05) and (D) up-regulates CRHR2 mRNA expression in SW620 and SW480 cells (**P < .01). (E) CRC specimens express higher IL-6 and IL-6R mRNA levels than controls. *P < .05. (F) Inhibition of IL-6-induced Stat3 (Tyr705) phosphorylation in CRHR2⁺ cells after stimulation with 20 ng/mL IL-6 and/or 0.1 μM Ucn2 for 30 minutes, as assessed by Western blot. Data shown are from a representative experiment. Lanes of bands shown have been cropped by the original whole gel images. (G) Validation of pStat3 (Tyr705) inhibition in SW620-CRHR2⁺ resected tumors from mice treated with Ucn2. Scale bar: 50 μm (scale bar proportional to the original microscope-derived image).

Figure 6

Corticotropin-releasing hormone receptor 2 (CRHR2)/urocortin-2 (Ucn2) signaling inhibits interleukin-6 (IL-6)-mediated colorectal cancer (CRC) growth, invasion, and migration. (A) CRHR2-dependant inhibition of IL-6-mediated cell proliferation of SW480-CRHR2⁺ cells. (B, C) CRHR2 specific inhibition of (B) migration and (C) invasion of SW620-CRHR2⁺ cells. *P ≤ .05; **P ≤ .01. (D) CRHR2 induction inhibits specifically the baseline and Ucn2-mediated invasion of the nonmetastatic cell line DLD1. *P < .05; **P < .01. (E) CRHR2-mediated inhibition of Ucn2-mediated migration of DLD1-CRHR2⁺ cells. **P < .01. In all assays CRHR2⁺ and EV CRC cells were treated with 20 ng/mL IL-6 alone or in combination with 0.1 μM Ucn2 ± 1 μM Ast2B for 72 hours for proliferation assessment or 48 hours for migration and invasion monitoring. DLD1 cells were pretreated with 20 ng/mL IL-6 for 72 hours before migration and invasion settings.

Figure 7

Corticotropin-releasing hormone receptor 2 (CRHR2)/urocortin-2 (Ucn2) signaling inhibits interleukin-6 (IL-6)-mediated colorectal cancer (CRC) growth and oncogenic epithelial-to-mesenchymal transition (EMT) through regulation of cell cycle- and EMT-related genes. (A) Heat map representing cell cycle–related gene array data derived by SW480-EV and SW480-CRHR2⁺ cells treated with 20 ng/mL IL-6 and 0.1 μM Ucn2 for 18 hours. Upper and lower panels: Down-regulation of cell cycle-promoting genes and up-regulation of cell cycle-suppressors in SW480-CRHR2⁺ cells, respectively. (B) Heat map representing EMT-related gene array data derived by SW620-EV and SW620-CRHR2⁺ cells treated with 20 ng/mL IL-6 and 0.1 μM Ucn2 for 18 hours. Upper and lower panels: Down-regulation of EMT-promoting genes and up-regulation of EMT-suppressors in SW620-CRHR2+ cells, respectively. (C) Validation of EMT inhibition at protein level in SW620-CRHR2+ protein extracts derived after cell treatment with 20 ng/mL IL-6 ± 0.1 μM Ucn2. Lanes of bands shown have been cropped by the original whole gel images.

Figure 8

Low corticotropin-releasing hormone receptor 2 (CRHR2) mRNA expression in human colorectal cancer (CRC) samples inversely correlates with (A) interleukin-6 (IL-6R) and (B) vimentin mRNA levels. (C) In contrast, a positive correlation was established between E-cadherin (CDH1) and high CRHR2 mRNA expression. Low and high CRHR2 expressions were set as detectable CRHR2 mRNA levels < mean ± standard error of the mean (SEM) or > mean ± SEM, respectively, calculated by the total CRC samples (n = 56). Among the 56 CRC samples tested, 3 were found with low CRHR2 expression, 11 with high expression, and the remaining 42 with undetectable expression. (D) Decreased expression of CRHR2 in CRC samples positively correlates with occurrence of distant metastasis. DM, distant metastasis (n = 18); NM, no metastasis (n = 21). (E) CRC patients with high tumoral CRHR2 mRNA expression (n = 11) have an increased 5-year survival rate compared with patients with low CRHR2 expression (n = 3).

Figure 9

Schematic representation of the corticotropin-releasing hormone receptor 2 (CRHR2)/urocortin-2 (Ucn2) targeted pathways and downstream events taking place after ectopic expression of CRHR2 in colorectal cancer (CRC) cells. (A) Constitutive activation of STAT3 in CRC by interleukin-6 (IL-6)/IL-6R signaling promotes expression of Stat3-targeted genes that regulate cell cycle and epithelial-to-mesenchymal transition (EMT). (B) CRHR2/Ucn2 signaling inhibits the expression of tumor-produced proinflammatory cytokines (PICs) such as IL-1b or IL-6 either via Ucn2 down-regulation or through a Ucn2-independent manner. CRHR2/Ucn2 also decreases the expression of IL-6R on the surface of CRC cells, thus resulting in attenuation of autocrine- or exocrine-mediated IL6R/IL6 signaling, which in turn leads to significant inhibition of Stat3 phosphorylation and its targeted genes. As constitutively active Stat3 is one of the major regulators of cell cycle and EMT in CRC cells, CRHR2/Ucn2-mediated Stat3 inhibition might facilitate at least in part the decreased proliferative, migratory, and invasive responses shown in CRHR2⁺ CRC cells. End, endogenous; Ex, exogenous.