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MMP3-mediated tumor progression is controlled transcriptionally by a novel IRF8-MMP3 interaction.

Banik D, Netherby CS, Bogner PN, Abrams SI - Oncotarget (2015)

Bottom Line: Importantly, the growth advantage due to IRF8-loss was significantly compromised after silencing MMP3 expression.Moreover, MMP3-loss reduced spontaneous lung metastasis in an orthotopic mouse model of mammary carcinoma.Thus, we identified a novel role of an IRF8-MMP3 axis in tumor progression, which unveils new therapeutic opportunities.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA.

ABSTRACT
Interferon regulatory factor-8 (IRF8), originally identified as a leukemic tumor suppressor, can also exert anti-neoplastic activities in solid tumors. We previously showed that IRF8-loss enhanced tumor growth, which was accompanied by reduced tumor-cell susceptibility to apoptosis. However, the impact of IRF8 expression on tumor growth could not be explained solely by its effects on regulating apoptotic response. Exploratory gene expression profiling further revealed an inverse relationship between IRF8 and MMP3 expression, implying additional intrinsic mechanisms by which IRF8 modulated neoplastic behavior. Although MMP3 expression was originally linked to tumor initiation, the role of MMP3 beyond this stage has remained unclear. Therefore, we hypothesized that MMP3 governed later stages of disease, including progression to metastasis, and did so through a novel IRF8-MMP3 axis. Altogether, we showed an inverse mechanistic relationship between IRF8 and MMP3 expression in tumor progression. Importantly, the growth advantage due to IRF8-loss was significantly compromised after silencing MMP3 expression. Moreover, MMP3-loss reduced spontaneous lung metastasis in an orthotopic mouse model of mammary carcinoma. MMP3 acted, in part, in a cell-intrinsic manner and served as a direct transcriptional target of IRF8. Thus, we identified a novel role of an IRF8-MMP3 axis in tumor progression, which unveils new therapeutic opportunities.

No MeSH data available.


Related in: MedlinePlus

IRF8 regulates MMP3 promoter activity in conjunction with PU.1A. Chromatin immunoprecipitation (ChIP) followed by RT-PCR on the indicated cell lines to determine binding of IRF8 and PU.1 to the putative consensus motif of the mouse MMP3 promoter (−1137 base pairs upstream of the TSS). Data represent one of two independent experiments. B. ChIP followed by quantitative PCR on the indicated cell line to determine enrichment of IRF8 or PU.1 at the putative consensus motif. C. Transfection assays using the CMS4 cell line to measure MMP3 promoter activity in the presence of cDNA encoding full-length IRF8, PU.1 cDNA or both (upper panel) vs. the corresponding empty vector control plasmids (lower panel). D. EMSA using CMS4-SC lysates after incubation with or without the indicated 32P-labeled oligonucleotide probe. Data are representative of two separate experiments.
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Figure 2: IRF8 regulates MMP3 promoter activity in conjunction with PU.1A. Chromatin immunoprecipitation (ChIP) followed by RT-PCR on the indicated cell lines to determine binding of IRF8 and PU.1 to the putative consensus motif of the mouse MMP3 promoter (−1137 base pairs upstream of the TSS). Data represent one of two independent experiments. B. ChIP followed by quantitative PCR on the indicated cell line to determine enrichment of IRF8 or PU.1 at the putative consensus motif. C. Transfection assays using the CMS4 cell line to measure MMP3 promoter activity in the presence of cDNA encoding full-length IRF8, PU.1 cDNA or both (upper panel) vs. the corresponding empty vector control plasmids (lower panel). D. EMSA using CMS4-SC lysates after incubation with or without the indicated 32P-labeled oligonucleotide probe. Data are representative of two separate experiments.

Mentions: To explore the possibility for a direct binding interaction between IRF8 and elements of the MMP3 promoter, we first visually inspected the murine MMP3 promoter in silico for IRF8 binding sites. In doing so, we identified a putative IRF8 binding site reflecting both ISRE (interferon-stimulated response element) and EICE (Ets/IRF composite element) motif characteristics [38] starting at position −1137 upstream from the transcriptional start site (GGAATGGAAA; Suppl. Fig. 1). ChIP assays were then performed using the IRF8-expressing or IRF8-deficient CMS4 cells. The protein-DNA complex was incubated with a ChIP-certified anti-IRF8 antibody, followed by PCR amplification of the MMP3 promoter region using primers surrounding a putative ISRE site located −1137 bp upstream from the transcription start site (Suppl. Fig. 1). Using the CMS4-SC cells, we found a direct binding interaction between IRF8 and the MMP3 promoter, based on the appearance of a PCR product reflecting the expected fragment size and subsequent quantification of these data which revealed a significant increase in the ChIP signal relative to the input DNA (Fig. 2A and 2B). Specificity for this IRF8-MMP3 axis was shown in two ways. First, no PCR product was detectable using the isotype control antibody or an antibody reactive against an unrelated transcription factor (i.e., pSTAT3) and, secondly, little to no PCR product was detectable in CMS4-IRF8lo cells (Fig. 2A). ChIP experiments were then performed using the 4T1 system and, as with the CMS4 system, a direct binding interaction was shown using 4T1-IRF8hi cells (Fig. 2A and 2B). An additional control included a ChIP-PCR reaction for an unrelated genomic region (i.e., GAPDH), which supported the integrity of the input DNA in these preparations.


MMP3-mediated tumor progression is controlled transcriptionally by a novel IRF8-MMP3 interaction.

Banik D, Netherby CS, Bogner PN, Abrams SI - Oncotarget (2015)

IRF8 regulates MMP3 promoter activity in conjunction with PU.1A. Chromatin immunoprecipitation (ChIP) followed by RT-PCR on the indicated cell lines to determine binding of IRF8 and PU.1 to the putative consensus motif of the mouse MMP3 promoter (−1137 base pairs upstream of the TSS). Data represent one of two independent experiments. B. ChIP followed by quantitative PCR on the indicated cell line to determine enrichment of IRF8 or PU.1 at the putative consensus motif. C. Transfection assays using the CMS4 cell line to measure MMP3 promoter activity in the presence of cDNA encoding full-length IRF8, PU.1 cDNA or both (upper panel) vs. the corresponding empty vector control plasmids (lower panel). D. EMSA using CMS4-SC lysates after incubation with or without the indicated 32P-labeled oligonucleotide probe. Data are representative of two separate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4558143&req=5

Figure 2: IRF8 regulates MMP3 promoter activity in conjunction with PU.1A. Chromatin immunoprecipitation (ChIP) followed by RT-PCR on the indicated cell lines to determine binding of IRF8 and PU.1 to the putative consensus motif of the mouse MMP3 promoter (−1137 base pairs upstream of the TSS). Data represent one of two independent experiments. B. ChIP followed by quantitative PCR on the indicated cell line to determine enrichment of IRF8 or PU.1 at the putative consensus motif. C. Transfection assays using the CMS4 cell line to measure MMP3 promoter activity in the presence of cDNA encoding full-length IRF8, PU.1 cDNA or both (upper panel) vs. the corresponding empty vector control plasmids (lower panel). D. EMSA using CMS4-SC lysates after incubation with or without the indicated 32P-labeled oligonucleotide probe. Data are representative of two separate experiments.
Mentions: To explore the possibility for a direct binding interaction between IRF8 and elements of the MMP3 promoter, we first visually inspected the murine MMP3 promoter in silico for IRF8 binding sites. In doing so, we identified a putative IRF8 binding site reflecting both ISRE (interferon-stimulated response element) and EICE (Ets/IRF composite element) motif characteristics [38] starting at position −1137 upstream from the transcriptional start site (GGAATGGAAA; Suppl. Fig. 1). ChIP assays were then performed using the IRF8-expressing or IRF8-deficient CMS4 cells. The protein-DNA complex was incubated with a ChIP-certified anti-IRF8 antibody, followed by PCR amplification of the MMP3 promoter region using primers surrounding a putative ISRE site located −1137 bp upstream from the transcription start site (Suppl. Fig. 1). Using the CMS4-SC cells, we found a direct binding interaction between IRF8 and the MMP3 promoter, based on the appearance of a PCR product reflecting the expected fragment size and subsequent quantification of these data which revealed a significant increase in the ChIP signal relative to the input DNA (Fig. 2A and 2B). Specificity for this IRF8-MMP3 axis was shown in two ways. First, no PCR product was detectable using the isotype control antibody or an antibody reactive against an unrelated transcription factor (i.e., pSTAT3) and, secondly, little to no PCR product was detectable in CMS4-IRF8lo cells (Fig. 2A). ChIP experiments were then performed using the 4T1 system and, as with the CMS4 system, a direct binding interaction was shown using 4T1-IRF8hi cells (Fig. 2A and 2B). An additional control included a ChIP-PCR reaction for an unrelated genomic region (i.e., GAPDH), which supported the integrity of the input DNA in these preparations.

Bottom Line: Importantly, the growth advantage due to IRF8-loss was significantly compromised after silencing MMP3 expression.Moreover, MMP3-loss reduced spontaneous lung metastasis in an orthotopic mouse model of mammary carcinoma.Thus, we identified a novel role of an IRF8-MMP3 axis in tumor progression, which unveils new therapeutic opportunities.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA.

ABSTRACT
Interferon regulatory factor-8 (IRF8), originally identified as a leukemic tumor suppressor, can also exert anti-neoplastic activities in solid tumors. We previously showed that IRF8-loss enhanced tumor growth, which was accompanied by reduced tumor-cell susceptibility to apoptosis. However, the impact of IRF8 expression on tumor growth could not be explained solely by its effects on regulating apoptotic response. Exploratory gene expression profiling further revealed an inverse relationship between IRF8 and MMP3 expression, implying additional intrinsic mechanisms by which IRF8 modulated neoplastic behavior. Although MMP3 expression was originally linked to tumor initiation, the role of MMP3 beyond this stage has remained unclear. Therefore, we hypothesized that MMP3 governed later stages of disease, including progression to metastasis, and did so through a novel IRF8-MMP3 axis. Altogether, we showed an inverse mechanistic relationship between IRF8 and MMP3 expression in tumor progression. Importantly, the growth advantage due to IRF8-loss was significantly compromised after silencing MMP3 expression. Moreover, MMP3-loss reduced spontaneous lung metastasis in an orthotopic mouse model of mammary carcinoma. MMP3 acted, in part, in a cell-intrinsic manner and served as a direct transcriptional target of IRF8. Thus, we identified a novel role of an IRF8-MMP3 axis in tumor progression, which unveils new therapeutic opportunities.

No MeSH data available.


Related in: MedlinePlus