Supplementary Material for: Intracellular Signaling Pathways Regulate Hormone-Dependent Kallikrein Gene Expression

<i>Objectives:</i> Our aim was to examine how certain signal transduction pathways influence the regulation of hormone-dependent kallikrein <i>(KLK) </i>gene expression in androgen-sensitive breast cancer cell lines. <i>Methods:</i> We used the breast cancer cell lines T47D and BT474, treated with steroid hormones or various pathway inhibitors. KLKs were quantified by ELISA. RT-PCR, Western blots and immunoprecipitations were used to assess transcript and protein levels. <i>Results:</i><i>PSA, KLK10, KLK11, KLK13</i> and <i>KLK14</i> are upregulated upon androgen stimulation in the T47D cell line. The expression of <i>PSA, KLK10 </i>and <i>KLK11 </i>was repressed by the MEK1/2 inhibitor U0126 and the PI3K inhibitor Wortmannin in the presence of the hormone, thus implicating the RAS/MEK/ERK and PI3K/AKT signaling pathways in regulating hormone-dependent <i>KLK</i> gene activation. Analysis of inhibitor-treated cells revealed changes in c-MYC expression with a pattern parallel to <i>KLK</i> gene expression. Chromatin immunoprecipitations identified androgen-dependent recruitment of specific transcription factors to the KLK proximal promoters, including c-MYC binding to <i>PSA</i> and <i>KLK11</i>. <i>Conclusion:</i> The hormone-specific upregulation of <i>PSA, KLK10</i> and <i>KLK11</i> in the breast cancer cell line T47D is dependent on major intracellular signaling pathways. This work provides a new dimension to the regulation of these cancer-related genes and the potential for new therapeutic targeting strategies.