Host Per2 clock gene mutation in the cancer host slows transplanted lung and colon Per2-replete tumor growth
Authors: Yin Xiong, Jovelyn Du-Quiton, Shaojin You, Masami Ohmori, Eun Y Oh, Patricia Ann Wood, Veronica Sue, William J. Hrushesky
Journal: Cancer Research (2006)

2526  Previous studies in our laboratory found an association between increased expression of the α2β1 integrin, a collagen and laminin receptor, and orderly regulated proliferation of epithelial cells. In vitro studies suggested that the lack of α2β1 integrin resulted in altered branching of mammary epithelial cells in 3-dimensional collagen gels. Based on these data we hypothesized that the α2β1 integrin was important for the normal development of the mammary gland and that mice lacking expression of the α2 integrin subunit (α2-null) would manifest defects in branching morphogenesis. The objective of the present study was to determine the role of the α2β1 integrin in the mouse mammary gland development using the α2-null mouse. To test our hypothesis mammary glands from virgin female α2-null and wild type (WT) mice were collected at 4-, 6-, 8-, 10- and 12 weeks of age. Total RNA was isolated from the second and third mammary gland and used for microarray and RT-PCR analysis. Mammary gland morphogenesis was evaluated using whole mount preparation of the fourth mammary gland stained with carmine aluminum. The architecture of the mammary gland including the number of branches and branch points was analyzed using Metamorph image software (Molecular Devices Corporation, PA). High magnification (20X) images were used to determine the average diameter of the ducts. Low magnification images (1X) were obtained to determine the total length of duct per breast, the branch points per length, the average length of the major segment and the average length of the branches. Mammary glands of α2-null mice showed a marked delay in penetration into the mammary fat pad at 4 and 6 weeks of age due to a 3-fold increase in epithelial cell apoptosis and a 2-fold decrease in epithelial cell proliferation. However, by 8-12 weeks the extent of penetration of the α2-null mammary gland into the fat pad was equivalent to that of the WT. In fact, the α2-null mammary branches showed increased tertiary branch points when compared with the WT mice. In addition, the diameter of the mammary ducts was 1.5-2 fold greater in the α2-null gland when compared to the WT at 8-12 weeks of age. To evaluate changes in gene expression that may contribute to the observed alterations in mammary gland development we evaluated the expression of other integrins and matrix metalloproteinases (MMP’s). RT-PCR analysis showed that there was no significant difference in the expression of the α3, α5, α6, αv, β1, β3, β4, and β5 integrin subunits or MMP-2, MMP-3, MMP-9 or MMP-14 between α2-null and WT mammary glands. In conclusion, α2-null mice demonstrated a delay in mammary gland growth at early stages of development, increased apoptosis and decreased Ki-67 expression at 4 weeks, and an increased of tertiary branch points and increased mammary duct diameter at 8-12 weeks of age. In addition, studies are ongoing to define the role of α2β1 integrin in breast cancer initiation, progression and metastasis.