Dmp1α Inhibits HER2/neu-Induced Mammary Tumorigenesis

<div><p>Our recent study shows a pivotal role of Dmp1 in quenching hyperproliferative signals from HER2 to the Arf-p53 pathway as a safety mechanism to prevent breast carcinogenesis. To directly demonstrate the role of Dmp1 in preventing HER2/neu-driven oncogenic transformation, we established <i>Flag-Dmp1α</i> transgenic mice (<i>MDTG</i>) under the control of the mouse mammary tumor virus (<i>MMTV</i>) promoter. The mice were viable but exhibited poorly developed mammary glands with markedly reduced milk production; thus more than half of parous females were unable to support the lives of new born pups. The mammary glands of the MDTG mice had very low Ki-67 expression but high levels of Arf, Ink4a, p53, and p21^Cip1, markers of senescence and accelerated aging. In all strains of generated <i>MDTG;neu</i> mice, tumor development was significantly delayed with decreased tumor weight. Tumors from <i>MDTG;neu</i> mice expressed Flag-Dmp1α and Ki-67 in a mutually exclusive fashion indicating that transgenic <i>Dmp1α</i> prevented tumor growth <i>in vivo</i>. Genomic DNA analyses showed that the <i>Dmp1α</i> transgene was partially lost in half of the <i>MDTG;neu</i> tumors, and Western blot analyses showed Dmp1α protein downregulation in 80% of the cases. Our data demonstrate critical roles of Dmp1 in preventing mammary tumorigenesis and raise the possibility of treating breast cancer by restoring Dmp1α expression.</p></div

Disease-free survival (DFS) curves of <i>MDTG;neu</i> mice in each strain.

<p>DFS curves for each strain of <i>MDTG;neu</i> females are shown. The <i>p</i> values were calculated in comparison to the survival of wild-type <i>MMTV-neu</i> mice (n = 25; medium survival, 197 days).</p

Immunohistochemical detection of Flag-Dmp1 and Ki67 in mammary glands of <i>MDTG</i> mice.

<p>A, B, C, and D. Immunohistochemical detection of the Flag-Dmp1 protein in nulliparous transgenic MMG. A) wild-type, B) <i>MDTG</i> strain 76, C) <i>MDTG</i> strain 79, and D) MDTG strain 138. E, F. Immunohistochemical detection of Ki67 in nulliparous MMG from wild-type (E) and <i>MDTG</i>, strain 76 (F) female. Note that Ki67 signals were barely detectable in MMG from a <i>MDTG</i> mouse.</p

Creation of <i>MMTV-Flag-Dmp1α</i> mice.

<p>A. The <i>Flag-Dmp1</i> cDNA (<i>Bam</i>HI fragment) was recovered from the pFLEX1-Dmp1 vector and was cloned into the <i>Hind</i>III site of the transgenic vector with <i>MMTV</i> promoter (<i>MMTV-SV40-BSSK</i>). B. Whole mammary gland mounts from 12-week-old nulliparous <i>MDTG</i> (strain 79) and wild-type mice. Scale bars show 2 mm. C. Photomicrographs of nulliparous and multi-parous female mammary glands (MMG) from wild-type and strain 76 <i>MMTV-Flag-Dmp1</i> (<i>MDTG</i>) mice (12-week-old). Wild-type mammary glands were well-dilated with milk production, which became more prominent after getting pregnant. <i>MDTG</i> mammary glands, on the other hand remained small, unopened with little milk production. These mammary glands did not open well with very low milk production even after getting pregnant, making it difficult to feed their pups. The scale bars show 100 µm.</p

Mutually exclusive expression of Flag-Dmp1 and Ki67 in <i>MDTG;neu</i> tumors.

<p>A. Total tumor weight harvested at sacrifice. The amount of tumors recovered at sacrifice (4 weeks after the first tumor was found) was significantly decreased in all three transgenic strains. B. Western blotting analyses of proteins expressed in <i>MDTG;neu</i> tumors. C. Mutually exclusive expression of Flag-Dmp1 and Ki67 in a <i>MDTG;neu</i> tumor (strain 76, #3987). Flag-Dmp1 was stained brown; Ki67 was stained blue. Scale bars show 100 µm. D. Gene copy number assay for <i>Flag-Dmp1</i> in paired tails and <i>MDTG;neu</i> tumors showing partial deletion of the transgene in half of the cases.</p

Immunohistochemical studies of <i>MDTG;neu</i> tumors.

<p>A. A <i>MDTG;neu</i> tumor (#3883) that shows areas of Flag-Dmp1 (brown) and islands of Dmp1-resistant tumor growth (white arrows). The Ki67-positive area is stained blue. B. Immunohistochemical staining of <i>MDTG;neu</i> tumor (#3987) with HER2 antibody. All of the tumor cells express the HER2/neu protein. Light green circles show areas of tumor growth while white circles show areas of tumor cell death. C. Double immunohistochemical staining of a <i>MDTG;neu</i> tumor (#3883) that shows significant overlap of Flag-Dmp1-positive cells (stained light brown in nuclei) and cleaved-caspase 3-positive cells (blue in the cytoplasm). A, B, C. Scale bars show 100 µm.</p

Immunohistochemical detection of p19^Arf, p16^Ink4a, p53, and p21^Cip1 in mammary glands of <i>MDTG</i> mice.

<p>All of these proteins were induced in MMG from <i>MDTG</i> females suggesting the activation of the p53 and Rb pathways by Dmp1 <i>in vivo</i>. Scale bars show 100 µm.</p