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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 tumor behavior in an MMP3-dependent mannerA. Knockdown of MMP3 expression in CMS4-IRF8lo cells compared to the SC population; data confirmed at RNA (qPCR, left) and protein (ELISA, right panel) levels. B. Tumor growth in wild-type mice (n = 16 per tumor cell line) or SCID mice (n = 5 per tumor cell line). C. Quantification of experimental lung nodules of the indicated tumor cell line following India ink staining. Each data point represents a single mouse. Representative lung-stained images are shown from each group. D. 4T1-IRF8hi or VC cells were orthotopically implanted into female wild-type BALB/c mice and tumor growth measured (n = 10 per tumor cell line). E. Lung-infiltrating tumor cells, assessed by clonogenic assays, using lung tissues from mice with similar primary tumor volumes (~1000 mm3). Each data point represents a single mouse. *P < 0.05.
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Figure 3: IRF8 regulates tumor behavior in an MMP3-dependent mannerA. Knockdown of MMP3 expression in CMS4-IRF8lo cells compared to the SC population; data confirmed at RNA (qPCR, left) and protein (ELISA, right panel) levels. B. Tumor growth in wild-type mice (n = 16 per tumor cell line) or SCID mice (n = 5 per tumor cell line). C. Quantification of experimental lung nodules of the indicated tumor cell line following India ink staining. Each data point represents a single mouse. Representative lung-stained images are shown from each group. D. 4T1-IRF8hi or VC cells were orthotopically implanted into female wild-type BALB/c mice and tumor growth measured (n = 10 per tumor cell line). E. Lung-infiltrating tumor cells, assessed by clonogenic assays, using lung tissues from mice with similar primary tumor volumes (~1000 mm3). Each data point represents a single mouse. *P < 0.05.

Mentions: Next, we examined whether manipulation of this IRF8-MMP3 interaction altered tumor growth in vivo. The approach taken was to silence MMP3 expression in the CMS4-IRF8lo cells. The prediction was that if the enhanced rate of tumor growth observed with CMS4-IRF8lo cells was due at least in part to higher MMP3 levels, then attenuating MMP3 expression altogether should reduce this IRF8-dependent growth advantage. Initial experiments analyzed knockdown efficiency of three separate shRNA-MMP3 constructs in the parental CMS4 cell line and identified two clones (clones #1 and #2) capable of significant gene-mediated silencing compared to the SC population (Suppl. Fig. 4). Based on knockdown efficiency, subsequent experiments were carried out with clone #1. Indeed, we found that decreased MMP3 expression in CMS4-IRF8lo cells (Fig. 3A; at RNA and protein levels) led to a significant reduction not only in local tumor growth, but also experimental lung metastasis (Fig. 3B & 3C; Suppl. Fig. 5 for tumor growth assay designs). The observation that CMS4-IRF8lo SC cells still grew faster than CMS4-IRF8loMMP3lo cells in SCID mice (Fig. 3B), which lack T and B cells, suggested that such differential tumor growth patterns can still occur without potential negative contributions of adaptive immune responses.


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 tumor behavior in an MMP3-dependent mannerA. Knockdown of MMP3 expression in CMS4-IRF8lo cells compared to the SC population; data confirmed at RNA (qPCR, left) and protein (ELISA, right panel) levels. B. Tumor growth in wild-type mice (n = 16 per tumor cell line) or SCID mice (n = 5 per tumor cell line). C. Quantification of experimental lung nodules of the indicated tumor cell line following India ink staining. Each data point represents a single mouse. Representative lung-stained images are shown from each group. D. 4T1-IRF8hi or VC cells were orthotopically implanted into female wild-type BALB/c mice and tumor growth measured (n = 10 per tumor cell line). E. Lung-infiltrating tumor cells, assessed by clonogenic assays, using lung tissues from mice with similar primary tumor volumes (~1000 mm3). Each data point represents a single mouse. *P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: IRF8 regulates tumor behavior in an MMP3-dependent mannerA. Knockdown of MMP3 expression in CMS4-IRF8lo cells compared to the SC population; data confirmed at RNA (qPCR, left) and protein (ELISA, right panel) levels. B. Tumor growth in wild-type mice (n = 16 per tumor cell line) or SCID mice (n = 5 per tumor cell line). C. Quantification of experimental lung nodules of the indicated tumor cell line following India ink staining. Each data point represents a single mouse. Representative lung-stained images are shown from each group. D. 4T1-IRF8hi or VC cells were orthotopically implanted into female wild-type BALB/c mice and tumor growth measured (n = 10 per tumor cell line). E. Lung-infiltrating tumor cells, assessed by clonogenic assays, using lung tissues from mice with similar primary tumor volumes (~1000 mm3). Each data point represents a single mouse. *P < 0.05.
Mentions: Next, we examined whether manipulation of this IRF8-MMP3 interaction altered tumor growth in vivo. The approach taken was to silence MMP3 expression in the CMS4-IRF8lo cells. The prediction was that if the enhanced rate of tumor growth observed with CMS4-IRF8lo cells was due at least in part to higher MMP3 levels, then attenuating MMP3 expression altogether should reduce this IRF8-dependent growth advantage. Initial experiments analyzed knockdown efficiency of three separate shRNA-MMP3 constructs in the parental CMS4 cell line and identified two clones (clones #1 and #2) capable of significant gene-mediated silencing compared to the SC population (Suppl. Fig. 4). Based on knockdown efficiency, subsequent experiments were carried out with clone #1. Indeed, we found that decreased MMP3 expression in CMS4-IRF8lo cells (Fig. 3A; at RNA and protein levels) led to a significant reduction not only in local tumor growth, but also experimental lung metastasis (Fig. 3B & 3C; Suppl. Fig. 5 for tumor growth assay designs). The observation that CMS4-IRF8lo SC cells still grew faster than CMS4-IRF8loMMP3lo cells in SCID mice (Fig. 3B), which lack T and B cells, suggested that such differential tumor growth patterns can still occur without potential negative contributions of adaptive immune responses.

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