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EVI1 promotes tumor growth via transcriptional repression of MS4A3.

Heller G, Rommer A, Steinleitner K, Etzler J, Hackl H, Heffeter P, Tomasich E, Filipits M, Steinmetz B, Topakian T, Klingenbrunner S, Ziegler B, Spittler A, Zöchbauer-Müller S, Berger W, Wieser R - J Hematol Oncol (2015)

Bottom Line: U937T_EVI1, a human myeloid cell line expressing EVI1 in a tetracycline regulable manner, was subjected to gene expression profiling. qRT-PCR was used to confirm the regulation of membrane-spanning-4-domains subfamily-A member-3 (MS4A3) by EVI1.The most strongly repressed gene was MS4A3, and its down-regulation by EVI1 was confirmed by qRT-PCR in additional, independent experimental model systems.MS4A3 mRNA levels were also negatively correlated with those of EVI1 in several published AML data sets.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine I, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria. gerwin.heller@meduniwien.ac.at.

ABSTRACT

Background: The transcription factor Ecotropic Virus Integration site 1 (EVI1) regulates cellular proliferation, differentiation, and apoptosis, and its overexpression contributes to an aggressive course of disease in myeloid leukemias and other malignancies. Notwithstanding, knowledge about the target genes mediating its biological and pathological functions remains limited. We therefore aimed to identify and characterize novel EVI1 target genes in human myeloid cells.

Methods: U937T_EVI1, a human myeloid cell line expressing EVI1 in a tetracycline regulable manner, was subjected to gene expression profiling. qRT-PCR was used to confirm the regulation of membrane-spanning-4-domains subfamily-A member-3 (MS4A3) by EVI1. Reporter constructs containing various parts of the MS4A3 upstream region were employed in luciferase assays, and binding of EVI1 to the MS4A3 promoter was investigated by chromatin immunoprecipitation. U937 derivative cell lines experimentally expressing EVI1 and/or MS4A3 were generated by retroviral transduction, and tested for their tumorigenicity by subcutaneous injection into severe combined immunodeficient mice.

Results: Gene expression microarray analysis identified 27 unique genes that were up-regulated, and 29 unique genes that were down-regulated, in response to EVI1 induction in the human myeloid cell line U937T. The most strongly repressed gene was MS4A3, and its down-regulation by EVI1 was confirmed by qRT-PCR in additional, independent experimental model systems. MS4A3 mRNA levels were also negatively correlated with those of EVI1 in several published AML data sets. Reporter gene assays and chromatin immunoprecipitation showed that EVI1 regulated MS4A3 via direct binding to a promoter proximal region. Experimental re-expression of MS4A3 in an EVI1 overexpressing cell line counteracted the tumor promoting effect of EVI1 in a murine xenograft model by increasing the rate of apoptosis.

Conclusions: Our data reveal MS4A3 as a novel direct target of EVI1 in human myeloid cells, and show that its repression plays a role in EVI1 mediated tumor aggressiveness.

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MS4A3 enhances apoptosis in EVI1-positive xenograft tumors. A) Whole sections of tumors derived from U937_vec_vec, U937_vec_MS4A3, U937_EVI1_vec, and U937_EVI1_MS4A3 cells were subjected to immunohistochemical staining for Ki-67 (left panel), or to staining for double strand breaks using the TUNEL method (right panel). Representative images are shown. Scale bar, 2 mm. B) Bar plot showing mean percentages + SEMs of TUNEL positive cells in 3 tumors of each of the 4 xenograft groups. *p < 0.05 (Student’s t-test, two-tailed).
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Fig5: MS4A3 enhances apoptosis in EVI1-positive xenograft tumors. A) Whole sections of tumors derived from U937_vec_vec, U937_vec_MS4A3, U937_EVI1_vec, and U937_EVI1_MS4A3 cells were subjected to immunohistochemical staining for Ki-67 (left panel), or to staining for double strand breaks using the TUNEL method (right panel). Representative images are shown. Scale bar, 2 mm. B) Bar plot showing mean percentages + SEMs of TUNEL positive cells in 3 tumors of each of the 4 xenograft groups. *p < 0.05 (Student’s t-test, two-tailed).

Mentions: Next, we asked whether EVI1 and/or MS4A3 would affect cellular proliferation in vitro or in vivo. Even though constitutive experimental expression of neither of these genes altered the cell cycle distribution of U937 cells in vitro in a significant manner (Figure 3A), EVI1 strongly and significantly enhanced the growth of tumors derived from these cells after subcutaneous injection into SCID mice, and re-expression of MS4A3 abolished this effect (Figure 3B). Immunohistochemical staining of tumor sections corroborated both the down-regulation of endogenous MS4A3 by EVI1 at the protein level, and the persistent expression of exogenous EVI1 and MS4A3 in the xenograft tumors (Figure 4). To investigate whether the observed disparity in tumor growth was attributable to different rates of proliferation and/or cell death, tumor sections were stained for the proliferation marker Ki-67 and for cell death-associated double strand breaks using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method. These experiments showed that all tumors contained large areas that were composed almost exclusively of Ki-67-positive cells, included high proportions of mitotic figures, and were interspersed only with sporadic TUNEL-positive cells (Figure 5A, Additional file 4: Figure S3A, and data not shown). On the other hand, some fractions of the tumors comprised high proportions, or consisted almost exclusively, of TUNEL-positive, Ki-67-negative cells (Figure 5A, Additional file 4: Figure S3B). The overall percentage of TUNEL positive cells was significantly higher in U937_EVI1_MS4A3 tumors than in U937_EVI1_vec tumors (Figure 5B), suggesting that re-expression of MS4A3 in EVI1-positive myeloid cells may slow tumor growth by enhancing the rate of cell death.Figure 3


EVI1 promotes tumor growth via transcriptional repression of MS4A3.

Heller G, Rommer A, Steinleitner K, Etzler J, Hackl H, Heffeter P, Tomasich E, Filipits M, Steinmetz B, Topakian T, Klingenbrunner S, Ziegler B, Spittler A, Zöchbauer-Müller S, Berger W, Wieser R - J Hematol Oncol (2015)

MS4A3 enhances apoptosis in EVI1-positive xenograft tumors. A) Whole sections of tumors derived from U937_vec_vec, U937_vec_MS4A3, U937_EVI1_vec, and U937_EVI1_MS4A3 cells were subjected to immunohistochemical staining for Ki-67 (left panel), or to staining for double strand breaks using the TUNEL method (right panel). Representative images are shown. Scale bar, 2 mm. B) Bar plot showing mean percentages + SEMs of TUNEL positive cells in 3 tumors of each of the 4 xenograft groups. *p < 0.05 (Student’s t-test, two-tailed).
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Fig5: MS4A3 enhances apoptosis in EVI1-positive xenograft tumors. A) Whole sections of tumors derived from U937_vec_vec, U937_vec_MS4A3, U937_EVI1_vec, and U937_EVI1_MS4A3 cells were subjected to immunohistochemical staining for Ki-67 (left panel), or to staining for double strand breaks using the TUNEL method (right panel). Representative images are shown. Scale bar, 2 mm. B) Bar plot showing mean percentages + SEMs of TUNEL positive cells in 3 tumors of each of the 4 xenograft groups. *p < 0.05 (Student’s t-test, two-tailed).
Mentions: Next, we asked whether EVI1 and/or MS4A3 would affect cellular proliferation in vitro or in vivo. Even though constitutive experimental expression of neither of these genes altered the cell cycle distribution of U937 cells in vitro in a significant manner (Figure 3A), EVI1 strongly and significantly enhanced the growth of tumors derived from these cells after subcutaneous injection into SCID mice, and re-expression of MS4A3 abolished this effect (Figure 3B). Immunohistochemical staining of tumor sections corroborated both the down-regulation of endogenous MS4A3 by EVI1 at the protein level, and the persistent expression of exogenous EVI1 and MS4A3 in the xenograft tumors (Figure 4). To investigate whether the observed disparity in tumor growth was attributable to different rates of proliferation and/or cell death, tumor sections were stained for the proliferation marker Ki-67 and for cell death-associated double strand breaks using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method. These experiments showed that all tumors contained large areas that were composed almost exclusively of Ki-67-positive cells, included high proportions of mitotic figures, and were interspersed only with sporadic TUNEL-positive cells (Figure 5A, Additional file 4: Figure S3A, and data not shown). On the other hand, some fractions of the tumors comprised high proportions, or consisted almost exclusively, of TUNEL-positive, Ki-67-negative cells (Figure 5A, Additional file 4: Figure S3B). The overall percentage of TUNEL positive cells was significantly higher in U937_EVI1_MS4A3 tumors than in U937_EVI1_vec tumors (Figure 5B), suggesting that re-expression of MS4A3 in EVI1-positive myeloid cells may slow tumor growth by enhancing the rate of cell death.Figure 3

Bottom Line: U937T_EVI1, a human myeloid cell line expressing EVI1 in a tetracycline regulable manner, was subjected to gene expression profiling. qRT-PCR was used to confirm the regulation of membrane-spanning-4-domains subfamily-A member-3 (MS4A3) by EVI1.The most strongly repressed gene was MS4A3, and its down-regulation by EVI1 was confirmed by qRT-PCR in additional, independent experimental model systems.MS4A3 mRNA levels were also negatively correlated with those of EVI1 in several published AML data sets.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine I, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria. gerwin.heller@meduniwien.ac.at.

ABSTRACT

Background: The transcription factor Ecotropic Virus Integration site 1 (EVI1) regulates cellular proliferation, differentiation, and apoptosis, and its overexpression contributes to an aggressive course of disease in myeloid leukemias and other malignancies. Notwithstanding, knowledge about the target genes mediating its biological and pathological functions remains limited. We therefore aimed to identify and characterize novel EVI1 target genes in human myeloid cells.

Methods: U937T_EVI1, a human myeloid cell line expressing EVI1 in a tetracycline regulable manner, was subjected to gene expression profiling. qRT-PCR was used to confirm the regulation of membrane-spanning-4-domains subfamily-A member-3 (MS4A3) by EVI1. Reporter constructs containing various parts of the MS4A3 upstream region were employed in luciferase assays, and binding of EVI1 to the MS4A3 promoter was investigated by chromatin immunoprecipitation. U937 derivative cell lines experimentally expressing EVI1 and/or MS4A3 were generated by retroviral transduction, and tested for their tumorigenicity by subcutaneous injection into severe combined immunodeficient mice.

Results: Gene expression microarray analysis identified 27 unique genes that were up-regulated, and 29 unique genes that were down-regulated, in response to EVI1 induction in the human myeloid cell line U937T. The most strongly repressed gene was MS4A3, and its down-regulation by EVI1 was confirmed by qRT-PCR in additional, independent experimental model systems. MS4A3 mRNA levels were also negatively correlated with those of EVI1 in several published AML data sets. Reporter gene assays and chromatin immunoprecipitation showed that EVI1 regulated MS4A3 via direct binding to a promoter proximal region. Experimental re-expression of MS4A3 in an EVI1 overexpressing cell line counteracted the tumor promoting effect of EVI1 in a murine xenograft model by increasing the rate of apoptosis.

Conclusions: Our data reveal MS4A3 as a novel direct target of EVI1 in human myeloid cells, and show that its repression plays a role in EVI1 mediated tumor aggressiveness.

Show MeSH
Related in: MedlinePlus