Expression of sonic hedgehog and correlation with aggressiveness in breast cancer.
Authors: FA Malik, A Sanders, L Ye, A Douglas-Jones, RE Mansel, WG Jiang
Journal: Cancer Research (2009)

Abstract Abstract #2055 Introduction&amp;#x2028; The Hedgehog pathway is an important regulatory pathway in the embryo development, particular in the development of mammary gland. It has been indicated to be a potential target in the therapies in cancers. Activation of the pathway has been shown to result in disease progression in certain solid tumours by possibly affecting the fate of cancer cells. The present study investigated the impact of modification of the expression of one of the Hedgehog pathway molecule, Sonic Hedgehog (SHH) on the cellular function in breast cancer and explored the expression pattern of SHH protein in human mammary tissues.&amp;#x2028; Materials and methods.&amp;#x2028; The study employed two human breast cancer cell lines, MCF-7 and MDA MB-231, both being positive in SHH expression. Three transgenes specifically targeting human Sonic Hedgehog (SHH) were constructed using an expression vector, each of the transgenes targeting different location of the SHH transcript. Stable tranfection strains were verified and established and designated as MCF7ΔSHH1/2/3 and MDA231ΔSHH1/2/3, respectively. Cellular functions in the wild type, control and SHH modified cells were analysed. Cell migration assay was based on a wounding assay using time lapse video recording. Matrix adhesion was assessed using a Matrigel coated culture surface. Invasiveness of the cells was determined using a transwell in vitro invasion assay. Cell growth assay was based on a colorimetric method. The presence and localisation of SHH protein in human mammary tissues were investigated using immunohistochemical method (n=32 pairs of normal and tumour tissues).&amp;#x2028; Results.&amp;#x2028; SHH transcripts were successfully knocked down by all three transgenes in both cell lines, as revealed by RT-PCR and quantitative RT-PCR analyses. Loss of SHH transcript in the cells lead to a significant reduction of migration using a wounding assay (migration distance over a 45 minute perid being 19.6±7.1µm for control cells and 1.6±7.6µm in MCF7ΔSHH cells, p=0.0035). Similarly, cells with reduced SHH expression displayed a significant reduction in their adherence to matrix protein (number of adherent cells for control MDA231 being 140.9±14.1 vs 80.8±13.0 for MDA231ΔSHH, p&amp;lt;0.001, MCF7 being 80.2±11.7 and for MCF7ΔSHH 41.2±12.6, p&amp;lt;0.001). The reduction of SHH was seen to be associated with significantly decreased invasiveness in both cells (p&amp;lt;0.01 when comparing control and transfected stains for both cells). In mammary tissues, SHH protein was seen to be faintly stained in the cytoplasmic region of normal epithelial cells. Little staining was seen in stromal cells. In tumour tissues, however, breast cancer cells showed a marked increase in the staining. Nucleus staining was occasionally observed.&amp;#x2028; In conclusion, SHH in breast cancer cells is correlated with the aggressiveness of the cells. Together with the increase expression of SHH protein in breast tumour tissues, SHH may present a therapeutic target in breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2055.