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. 2015 Nov 10;6(35):36928-42.
doi: 10.18632/oncotarget.6047.

MDA-7/IL-24 functions as a tumor suppressor gene in vivo in transgenic mouse models of breast cancer

Mitchell E Menezes  1 , Xue-Ning Shen  1 , Swadesh K Das  1   2   3 , Luni Emdad  1   2   3 , Chunqing Guo  1 , Fang Yuan  1 , You-Jun Li  4 , Michael C Archer  5   6 , Eldad Zacksenhaus  5   7 , Jolene J Windle  1   2   3 , Mark A Subler  1 , Yaacov Ben-David  5   8 , Devanand Sarkar  1   2   3 , Xiang-Yang Wang  1   2   3 , Paul B Fisher  1   2   3
Affiliations

MDA-7/IL-24 functions as a tumor suppressor gene in vivo in transgenic mouse models of breast cancer

Mitchell E Menezes et al. Oncotarget. .

Abstract

Melanoma differentiation associated gene-7/Interleukin-24 (MDA-7/IL-24) is a novel member of the IL-10 gene family that selectively induces apoptosis and toxic autophagy in a broad spectrum of human cancers, including breast cancer, without harming normal cells or tissues. The ability to investigate the critical events underlying cancer initiation and progression, as well as the capacity to test the efficacy of novel therapeutics, has been significantly advanced by the development of genetically engineered mice (GEMs) that accurately recapitulate specific human cancers. We utilized three transgenic mouse models to better comprehend the in vivo role of MDA-7/IL-24 in breast cancer. Using the MMTV-PyMT spontaneous mammary tumor model, we confirmed that exogenously introducing MDA-7/IL-24 using a Cancer Terminator Virus caused a reduction in tumor burden and also produced an antitumor "bystander" effect. Next we performed xenograft studies in a newly created MMTV-MDA-7 transgenic model that over-expresses MDA-7/IL-24 in the mammary glands during pregnancy and lactation, and found that MDA-7/IL-24 overexpression delayed tumor growth following orthotopic injection of a murine PDX tumor cell line (mPDX) derived from a tumor formed in an MMTV-PyMT mouse. We also crossed the MMTV-MDA-7 line to MMTV-Erbb2 transgenic mice and found that MDA-7/IL-24 overexpression delayed the onset of mammary tumor development in this model of spontaneous mammary tumorigenesis as well. Finally, we assessed the role of MDA-7/IL-24 in immune regulation, which can potentially contribute to tumor suppression in vivo. Our findings provide further direct in vivo evidence for the role of MDA-7/IL-24 in tumor suppression in breast cancer in immune-competent transgenic mice.

Keywords: MMTV-MDA-7 mice; MMTV-MDA-7/MMTV-Erbb2 mice; MMTV-PyMT mice; melanoma differentiation associated gene-7/interleukin-24 (MDA-7/IL-24); transgenic mice.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. MDA-7/IL-24 inhibits tumor growth in MMTV-PyMT transgenic mice
A. Mice treated intratumorally with Ad5-E1A, Ad5-CTV or untreated controls were monitored for tumor burden over 4 weeks following first appearance of tumors. Tumor burden is represented in arbitrary units. B. Fold-change in total tumor volume in mice receiving Ad5-E1A, Ad5-CTV and control untreated mice. C. Fold-change in total tumor weight in mice receiving Ad5-E1A, Ad5-CTV and control untreated mice. D. Representative images of total tumors present in MMTV-PyMT mice treated with Ad5-E1A, Ad5-CTV and untreated controls at the time of sacrifice. Image dimensions are approximately adjusted to match the scale in each image. E. Comparison of tumor burden in control untreated mice (tumors from left and right sides) and Ad5-CTV-treated mice (injected and uninjected tumors). Uninjected tumors in mice receiving Ad5-CTV also showed a decrease in tumor size, indicating an anti-tumor “bystander” activity. *, p < 0.05; **, p < 0.01; NS, not significant.
Figure 2
Figure 2. Ad5-CTV leads to robust expression of MDA-7/IL-24 in Ad5-CTV injected tumors in MMTV-PyMT mice
Ad5-E1A, Ad5-CTV and control untreated tumors were sectioned and immunohistochemistry was performed for H&E staining and to assess MDA-7/IL-24 expression. As would be expected, tumors injected with Ad5-CTV showed MDA-7/IL-24 expression. Yellow bars = 100 μm, white bars = 20 μm.
Figure 3
Figure 3. MDA-7/IL-24 is robustly expressed in MMTV-MDA-7 transgenic mice during pregnancy and lactation
Mammary fat pads were harvested from MMTV-MDA-7 (positive and negative/control/non-transgenic littermate) pregnant, and lactating mice and protein and RNA were extracted. MDA-7/IL-24 expression was assessed in the mammary fat pad of MMTV-MDA-7 transgenic mice at the transcript A. and protein B. levels. C. Tumor cells were harvested from MMTV-PyMT mice bearing tumors and grown in vitro. These cells were stably transfected with a luciferase-expressing construct to generate MMTV-PyMT luc cells. Luciferase expression was assessed in these MMTV-PyMT luc (mPDX luc) cells. D. Schematic of the xenograft study in MMTV-MDA-7 transgenic mice. Female (MMTV-MDA-7 positive and negative/control/non-transgenic littermates) and male mice were housed together to allow for offspring. Following the birth of first litter, MMTV-PyMT luc cells were injected into the 4th mammary fat pad of the female mice and tumor growth was monitored by bioluminescent imaging.
Figure 4
Figure 4. MDA-7/IL-24 suppresses tumor growth in MMTV-MDA-7 transgenic mice
A. Representative bioluminescent images at 4, 8 and 10-weeks post injection of MMTV-PyMT luc (mPDX luc) cells into the 4th mammary fat pad of female MMTV-MDA-7 negative (control) and positive mice. B. Graphical representation of the quantification of luminescence signals. IVIS spectrum coupled with Living Image 4.3.1 was used for the quantification. MMTV-MDA-7 mice showed no detectable luminescence signal when imaged alongside the control non-transgenic mice, (N.D. = not detected). C. Images of tumors from MMTV-MDA-7 negative (control/non-transgenic littermate) and positive mice at sacrifice. D. Immunohistochemistry showing expression of MDA-7/IL-24 in tumor sections of MMTV-MDA-7 positive but not MMTV-MDA-7 negative (control/non-transgenic littermate) mice. Yellow bars = 100 μm, white bars = 20 μm.
Figure 5
Figure 5. Tumor onset is delayed in MMTV-MDA-7/MMTV-Erbb2 compound transgenic mice
A. Schematic of generation of MMTV-MDA-7/MMTV-Erbb2 compound transgenic mice. MMTV-MDA-7 hemizygous mice were mated with MMTV-Erbb2 homozygous mice. B. Kaplan Meier analysis showing delay in tumor onset in MMTV-MDA-7/MMTV-Erbb2 compound transgenic mice. C. Immunohistochemistry showing H&E staining and MDA-7/IL-24 expression in tumor sections of MMTV-Erbb2 and MMTV-MDA-7/MMTV-Erbb2 mice. Yellow bars = 100 μm, white bars = 20 μm. ***, p < 0.001.
Figure 6
Figure 6. MDA-7/IL-24 enhances anti-tumor immune responses against MMTV-PyMT mammary tumors
Mammary tumors of MMTV-PyMT transgenic mice were treated with Ad5-CTV or Ad5-E1A as a control. A. and B. CD8+ and CD4+ T cell infiltration in the treated tumors and non-treated tumors were analyzed by FACS. The frequency in panel A is calculated as the ratio of CD8+ or CD4+ T cells among all the cells in the tumors. C. to E. Frequency of IFN-γ or granzyme B producing CD8+ T cells was assayed by intracellular IFN-γ staining and FACS. The numbers in panel C are the frequency of IFN-γ producing cells among CD8+ or CD4+ T cells. *, p < 0.05; **, p < 0.01; NS, not significant.
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