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Interleukin 6 downregulates p53 expression and activity by stimulating ribosome biogenesis: a new pathway connecting inflammation to cancer.

Brighenti E, Calabrese C, Liguori G, Giannone FA, Trerè D, Montanaro L, Derenzini M - Oncogene (2014)

Bottom Line: The p53 downregulation induced the acquisition of cellular phenotypic changes characteristic of epithelial-mesenchymal transition, such as a reduced level of E-cadherin expression, increased cell invasiveness and a decreased response to cytotoxic stresses.We found that these changes also occurred in colon epithelial cells of patients with ulcerative colitis, a very representative example of chronic inflammation at high risk for tumor development.Taken together, the present results highlight a new mechanism that may link chronic inflammation to cancer, based on p53 downregulation, which is activated by the enhancement of rRNA transcription upon IL-6 exposure.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental, Diagnostic and Specialty Medicine, Bologna University, Bologna, Italy.

ABSTRACT
Chronic inflammation is an established risk factor for the onset of cancer, and the inflammatory cytokine IL-6 has a role in tumorigenesis by enhancing proliferation and hindering apoptosis. As factors stimulating proliferation also downregulate p53 expression by enhancing ribosome biogenesis, we hypothesized that IL-6 may cause similar changes in inflamed tissues, thus activating a mechanism that favors neoplastic transformation. Here, we showed that IL-6 downregulated the expression and activity of p53 in transformed and untransformed human cell lines. This was the consequence of IL-6-dependent stimulation of c-MYC mRNA translation, which was responsible for the upregulation of rRNA transcription. The enhanced rRNA transcription stimulated the MDM2-mediated proteasomal degradation of p53, by reducing the availability of ribosome proteins for MDM2 binding. The p53 downregulation induced the acquisition of cellular phenotypic changes characteristic of epithelial-mesenchymal transition, such as a reduced level of E-cadherin expression, increased cell invasiveness and a decreased response to cytotoxic stresses. We found that these changes also occurred in colon epithelial cells of patients with ulcerative colitis, a very representative example of chronic inflammation at high risk for tumor development. Histochemical and immunohistochemical analysis of colon biopsy samples showed an upregulation of ribosome biogenesis, a reduced expression of p53, together with a focal reduction or absence of E-cadherin expression in chronic colitis in comparison with normal mucosa samples. These changes disappeared after treatment with anti-inflammatory drugs. Taken together, the present results highlight a new mechanism that may link chronic inflammation to cancer, based on p53 downregulation, which is activated by the enhancement of rRNA transcription upon IL-6 exposure.

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Related in: MedlinePlus

IL-6 treatment stimulates rRNA transcription by activation of c-myc protein in NCM460 and HepG2 cell lines. (a) Real-time–PCR analysis of the 45S rRNA expression in NCM460, HepG2, SW1990 and LS174T cells after 24 h of IL-6 treatment performed with a dose of 50 ng/ml. (b) Visualization of rRNA synthesis in control and IL-6-treated NCM460 and HepG2 cells. Cells were labeled with 5-FU for 15 min, and 5-FU revealed by specific FITC-conjugated monoclonal antibody. DAPI counterstaining. Scale bar=20 μm. (c) Representative western blot and densitometric analysis of c-myc expression in NCM460 and HepG2 cells treated with IL-6 for 1 and 3 h. (d) Real-time–PCR analysis of the c-MYC mRNA levels in NCM460 and HepG2 after 1 and 3 h of IL-6 treatment. (e) IRES-mediated translation assessed by measuring the FLuc and RLuc activity in control and 4 h IL-6-treated NCM460 and HepG2 cells after 8 h transfection with a bicistronic mRNA transcribed either from pRF (top) or from pR-c-MYC-IRES-F (bottom). (f) Time-course analysis of c-myc protein expression in control and 24 h-IL-6-stimulated HepG2 cells, exposed to cycloheximide (CHX) at a concentration of 20 μg/ml. (g) Real-time–PCR and western blot analysis of c-MYC expression in scrambled control siRNA (SCR) and in 24 h c-MYC-silenced (MYC−) HepG2 cells exposed to IL-6 for 24 h. The right panel shows the 45S rRNA expression in the same experimental condition. Histograms show the values (mean±s.d.) of three experiments. *P<0.05; **P<0.01. n.s., not significant.
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fig1: IL-6 treatment stimulates rRNA transcription by activation of c-myc protein in NCM460 and HepG2 cell lines. (a) Real-time–PCR analysis of the 45S rRNA expression in NCM460, HepG2, SW1990 and LS174T cells after 24 h of IL-6 treatment performed with a dose of 50 ng/ml. (b) Visualization of rRNA synthesis in control and IL-6-treated NCM460 and HepG2 cells. Cells were labeled with 5-FU for 15 min, and 5-FU revealed by specific FITC-conjugated monoclonal antibody. DAPI counterstaining. Scale bar=20 μm. (c) Representative western blot and densitometric analysis of c-myc expression in NCM460 and HepG2 cells treated with IL-6 for 1 and 3 h. (d) Real-time–PCR analysis of the c-MYC mRNA levels in NCM460 and HepG2 after 1 and 3 h of IL-6 treatment. (e) IRES-mediated translation assessed by measuring the FLuc and RLuc activity in control and 4 h IL-6-treated NCM460 and HepG2 cells after 8 h transfection with a bicistronic mRNA transcribed either from pRF (top) or from pR-c-MYC-IRES-F (bottom). (f) Time-course analysis of c-myc protein expression in control and 24 h-IL-6-stimulated HepG2 cells, exposed to cycloheximide (CHX) at a concentration of 20 μg/ml. (g) Real-time–PCR and western blot analysis of c-MYC expression in scrambled control siRNA (SCR) and in 24 h c-MYC-silenced (MYC−) HepG2 cells exposed to IL-6 for 24 h. The right panel shows the 45S rRNA expression in the same experimental condition. Histograms show the values (mean±s.d.) of three experiments. *P<0.05; **P<0.01. n.s., not significant.

Mentions: We analyzed the effect of IL-6 on ribosome biogenesis in the following four human epithelial cell lines: one from normal colon epithelium (NCM460 cell line), one from colon carcinoma (LS174T cell line), one from hepatocellular carcinoma (HepG2 cell line) and one from pancreas carcinoma (SW1990 cell line). The changes in rRNA transcriptional activity in these four cell lines exposed to IL-6 were ascertained using real-time–PCR analysis of the 45S prerRNA expression (Figure 1a). The rRNA synthesis was also evaluated by the immunofluorescence detection of nucleolar 5-fluorouridine (5-FU) incorporation into nascent rRNA in NCM460 and HepG2 cell lines (Figure 1b). Both methodological approaches revealed that IL-6 greatly enhanced rRNA transcription. As these data indicate that IL-6 enhances c-myc expression16, 17 in order to study the mechanism involved in the IL-6 stimulation of rRNA transcription, we investigated whether IL-6 enhanced the expression of c-myc protein also in the human epithelial cells used in the present study. For this reason we exposed both the NCM460 and the HepG2 cell lines to IL-6. We found that IL-6 significantly increased c-myc protein expression as early as 1 h after IL-6 stimulation in both cell lines, as evaluated by western blot analysis (Figure 1c). Unlike c-myc protein expression, the level of c-MYC mRNA did not change for up to 3 h after IL-6 exposure (Figure 1d). This suggested that, in our experimental conditions, a post-transcriptional mechanism was activated by IL-6 and was responsible for the increase in the c-myc protein. IL-6 has been reported to control the c-myc protein level either by increasing STAT3-mediated mRNA c-MYC transcription16 or via a stimulatory effect on the c-MYC internal ribosome entry site.17 Therefore, we analyzed the effect of IL-6 exposure on c-MYC mRNA IRES-dependent translation. For this purpose, NCM460 and HepG2 cells were transfected with a bicistronic in vitro transcribed mRNA in which the c-MYC-IRES sequence was inserted between two reporter luciferase cistrons (Renilla and Firefly, Promega, Milan, Italy). Evaluation of the ratio between the Firefly and Renilla activities indicated the IRES-dependent translation rate. We found that IL-6 treatment strongly stimulated the translation initiation mediated by the c-MYC-IRES in NCM460 and HepG2 cells (Figure 1e). As a control, a similar bicistronic transcript not containing the c-MYC-IRES element was transfected in IL-6-treated and control cells. In this case, no differences in the ratio between the Firefly and Renilla activities were detected. These findings indicated that the increased expression of c-myc protein observed in both cell lines after IL-6 exposure was due to an enhanced c-MYC mRNA IRES-dependent translation. In order to exclude that other post-transcriptional mechanisms may be involved, such as changes in protein stability, we evaluated the c-myc protein half-life in IL-6-stimulated HepG2 cells after protein synthesis inhibition by cycloheximide at a dose capable of completely inhibiting protein synthesis. We found that the half-life of c-myc protein was unchanged after IL-6 treatment (Figure 1f).


Interleukin 6 downregulates p53 expression and activity by stimulating ribosome biogenesis: a new pathway connecting inflammation to cancer.

Brighenti E, Calabrese C, Liguori G, Giannone FA, Trerè D, Montanaro L, Derenzini M - Oncogene (2014)

IL-6 treatment stimulates rRNA transcription by activation of c-myc protein in NCM460 and HepG2 cell lines. (a) Real-time–PCR analysis of the 45S rRNA expression in NCM460, HepG2, SW1990 and LS174T cells after 24 h of IL-6 treatment performed with a dose of 50 ng/ml. (b) Visualization of rRNA synthesis in control and IL-6-treated NCM460 and HepG2 cells. Cells were labeled with 5-FU for 15 min, and 5-FU revealed by specific FITC-conjugated monoclonal antibody. DAPI counterstaining. Scale bar=20 μm. (c) Representative western blot and densitometric analysis of c-myc expression in NCM460 and HepG2 cells treated with IL-6 for 1 and 3 h. (d) Real-time–PCR analysis of the c-MYC mRNA levels in NCM460 and HepG2 after 1 and 3 h of IL-6 treatment. (e) IRES-mediated translation assessed by measuring the FLuc and RLuc activity in control and 4 h IL-6-treated NCM460 and HepG2 cells after 8 h transfection with a bicistronic mRNA transcribed either from pRF (top) or from pR-c-MYC-IRES-F (bottom). (f) Time-course analysis of c-myc protein expression in control and 24 h-IL-6-stimulated HepG2 cells, exposed to cycloheximide (CHX) at a concentration of 20 μg/ml. (g) Real-time–PCR and western blot analysis of c-MYC expression in scrambled control siRNA (SCR) and in 24 h c-MYC-silenced (MYC−) HepG2 cells exposed to IL-6 for 24 h. The right panel shows the 45S rRNA expression in the same experimental condition. Histograms show the values (mean±s.d.) of three experiments. *P<0.05; **P<0.01. n.s., not significant.
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fig1: IL-6 treatment stimulates rRNA transcription by activation of c-myc protein in NCM460 and HepG2 cell lines. (a) Real-time–PCR analysis of the 45S rRNA expression in NCM460, HepG2, SW1990 and LS174T cells after 24 h of IL-6 treatment performed with a dose of 50 ng/ml. (b) Visualization of rRNA synthesis in control and IL-6-treated NCM460 and HepG2 cells. Cells were labeled with 5-FU for 15 min, and 5-FU revealed by specific FITC-conjugated monoclonal antibody. DAPI counterstaining. Scale bar=20 μm. (c) Representative western blot and densitometric analysis of c-myc expression in NCM460 and HepG2 cells treated with IL-6 for 1 and 3 h. (d) Real-time–PCR analysis of the c-MYC mRNA levels in NCM460 and HepG2 after 1 and 3 h of IL-6 treatment. (e) IRES-mediated translation assessed by measuring the FLuc and RLuc activity in control and 4 h IL-6-treated NCM460 and HepG2 cells after 8 h transfection with a bicistronic mRNA transcribed either from pRF (top) or from pR-c-MYC-IRES-F (bottom). (f) Time-course analysis of c-myc protein expression in control and 24 h-IL-6-stimulated HepG2 cells, exposed to cycloheximide (CHX) at a concentration of 20 μg/ml. (g) Real-time–PCR and western blot analysis of c-MYC expression in scrambled control siRNA (SCR) and in 24 h c-MYC-silenced (MYC−) HepG2 cells exposed to IL-6 for 24 h. The right panel shows the 45S rRNA expression in the same experimental condition. Histograms show the values (mean±s.d.) of three experiments. *P<0.05; **P<0.01. n.s., not significant.
Mentions: We analyzed the effect of IL-6 on ribosome biogenesis in the following four human epithelial cell lines: one from normal colon epithelium (NCM460 cell line), one from colon carcinoma (LS174T cell line), one from hepatocellular carcinoma (HepG2 cell line) and one from pancreas carcinoma (SW1990 cell line). The changes in rRNA transcriptional activity in these four cell lines exposed to IL-6 were ascertained using real-time–PCR analysis of the 45S prerRNA expression (Figure 1a). The rRNA synthesis was also evaluated by the immunofluorescence detection of nucleolar 5-fluorouridine (5-FU) incorporation into nascent rRNA in NCM460 and HepG2 cell lines (Figure 1b). Both methodological approaches revealed that IL-6 greatly enhanced rRNA transcription. As these data indicate that IL-6 enhances c-myc expression16, 17 in order to study the mechanism involved in the IL-6 stimulation of rRNA transcription, we investigated whether IL-6 enhanced the expression of c-myc protein also in the human epithelial cells used in the present study. For this reason we exposed both the NCM460 and the HepG2 cell lines to IL-6. We found that IL-6 significantly increased c-myc protein expression as early as 1 h after IL-6 stimulation in both cell lines, as evaluated by western blot analysis (Figure 1c). Unlike c-myc protein expression, the level of c-MYC mRNA did not change for up to 3 h after IL-6 exposure (Figure 1d). This suggested that, in our experimental conditions, a post-transcriptional mechanism was activated by IL-6 and was responsible for the increase in the c-myc protein. IL-6 has been reported to control the c-myc protein level either by increasing STAT3-mediated mRNA c-MYC transcription16 or via a stimulatory effect on the c-MYC internal ribosome entry site.17 Therefore, we analyzed the effect of IL-6 exposure on c-MYC mRNA IRES-dependent translation. For this purpose, NCM460 and HepG2 cells were transfected with a bicistronic in vitro transcribed mRNA in which the c-MYC-IRES sequence was inserted between two reporter luciferase cistrons (Renilla and Firefly, Promega, Milan, Italy). Evaluation of the ratio between the Firefly and Renilla activities indicated the IRES-dependent translation rate. We found that IL-6 treatment strongly stimulated the translation initiation mediated by the c-MYC-IRES in NCM460 and HepG2 cells (Figure 1e). As a control, a similar bicistronic transcript not containing the c-MYC-IRES element was transfected in IL-6-treated and control cells. In this case, no differences in the ratio between the Firefly and Renilla activities were detected. These findings indicated that the increased expression of c-myc protein observed in both cell lines after IL-6 exposure was due to an enhanced c-MYC mRNA IRES-dependent translation. In order to exclude that other post-transcriptional mechanisms may be involved, such as changes in protein stability, we evaluated the c-myc protein half-life in IL-6-stimulated HepG2 cells after protein synthesis inhibition by cycloheximide at a dose capable of completely inhibiting protein synthesis. We found that the half-life of c-myc protein was unchanged after IL-6 treatment (Figure 1f).

Bottom Line: The p53 downregulation induced the acquisition of cellular phenotypic changes characteristic of epithelial-mesenchymal transition, such as a reduced level of E-cadherin expression, increased cell invasiveness and a decreased response to cytotoxic stresses.We found that these changes also occurred in colon epithelial cells of patients with ulcerative colitis, a very representative example of chronic inflammation at high risk for tumor development.Taken together, the present results highlight a new mechanism that may link chronic inflammation to cancer, based on p53 downregulation, which is activated by the enhancement of rRNA transcription upon IL-6 exposure.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental, Diagnostic and Specialty Medicine, Bologna University, Bologna, Italy.

ABSTRACT
Chronic inflammation is an established risk factor for the onset of cancer, and the inflammatory cytokine IL-6 has a role in tumorigenesis by enhancing proliferation and hindering apoptosis. As factors stimulating proliferation also downregulate p53 expression by enhancing ribosome biogenesis, we hypothesized that IL-6 may cause similar changes in inflamed tissues, thus activating a mechanism that favors neoplastic transformation. Here, we showed that IL-6 downregulated the expression and activity of p53 in transformed and untransformed human cell lines. This was the consequence of IL-6-dependent stimulation of c-MYC mRNA translation, which was responsible for the upregulation of rRNA transcription. The enhanced rRNA transcription stimulated the MDM2-mediated proteasomal degradation of p53, by reducing the availability of ribosome proteins for MDM2 binding. The p53 downregulation induced the acquisition of cellular phenotypic changes characteristic of epithelial-mesenchymal transition, such as a reduced level of E-cadherin expression, increased cell invasiveness and a decreased response to cytotoxic stresses. We found that these changes also occurred in colon epithelial cells of patients with ulcerative colitis, a very representative example of chronic inflammation at high risk for tumor development. Histochemical and immunohistochemical analysis of colon biopsy samples showed an upregulation of ribosome biogenesis, a reduced expression of p53, together with a focal reduction or absence of E-cadherin expression in chronic colitis in comparison with normal mucosa samples. These changes disappeared after treatment with anti-inflammatory drugs. Taken together, the present results highlight a new mechanism that may link chronic inflammation to cancer, based on p53 downregulation, which is activated by the enhancement of rRNA transcription upon IL-6 exposure.

Show MeSH
Related in: MedlinePlus