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Hh-Gli signaling pathway activity in ER-positive (MCF-7) and ER-negative (Sk-Br-3) breast cancer cell lines treated with tamoxifen

Hedgehog-Gli (Hh-Gli) signaling pathway regulates a wide variety of processes during embryonic development and adult tissue homeostasis, and its malfunctioning is often associated with tumorigenesis and developmental malformations. Activation of Hh-Gli signaling has been reported in about 30% of human cancers including breast cancer. The ligand protein Sonic Hedhegog (Shh) binds to the transmembrane receptor Patched (Ptch). Ptch then releases its inhibition of Smoothened (Smo), and a cytoplasmatic cascade of phosphorylation and dephosphorylation events leading to activation of transcription factor Gli and transcription of target genes, which include Cyclin D, Cyclin E, members of Wnt and TGFβ signaling pathways, and Ptch itself. Tamoxifen inhibits proliferation and induces apoptosis of breast cancer cells by estrogen receptor (ER)-dependent modulation of gene expression. In this work we focus our interest on human breast cancer cell lines, ER-positive (MCF-7) and ER-negative (Sk-Br-3), where we intend to see how Hh-Gli signaling pathway is expressed in cell lines treated with tamoxifen. Also, we are interested in potential therapeutical application of a known pathway inhibitor cyclopamine in combination with tamoxifen, whose activities we intend to test on these cells. Using MTT assay to determine cell viability we observed that tamoxifen induced rapid death in both ER-positive (MCF-7) and ER-negative (Sk-Br-3) breast cancer cell lines. Combined treatment with cyclopamine and tamoxifen differred between the tested cell lines, and the reactivity to these drugs is dependant on the order in which they are administered. Real-Time PCR demonstrated variable expression of the Hh-Gli pathway genes (SHH, PTCH, GLI1, SMO and SUFU). Western blot analysis shows difference in protein expression of main members of the pathway (Ptch, Smo, Shh and Gli1). Our preliminary results suggest Hh-Gli pathway involvement in tumorigenesis in tested cancer cell lines with variable intensity and that tamoxifen resulted rapid cell death in both investigated cancer cell lines, alone or in combination with cyclopamine. Those results gave a promising model for further studies as an effective strategy for cancer treatment.

Hh-Gli signaling pathway ; breast cancer ; tamoxifen ; cyclopamine

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