Inhibition of the Sonic Hedgehog Pathway using Small Molecule Inhibitors: Targeting Colon Cancer Stem Cells

Abstract

Background: The Hedgehog (HH) pathway is a key regulator of many important processes in vertebrate embryonic development, including stem cell maintenance, cell differentiation, tissue polarity and cell proliferation. During pathway activation, Ptch no longer inhibits Smo and the full length Gli translocates to the nucleus resulting in the transcription of oncogenes. When constitutively activated, this leads to tumorigenesis in several human cancers. Cyclopamine acts as an antagonist of the HH signalling pathway by directly binding to the Smo heptahelical domain. The involvement of this pathway in metastasis, and its presence in cancer stem cells (CSCs), makes it a valid option for developing a targeted therapeutic against it.

Methods: CSC were isolated from DLD1 and HT29 cell lines using magnetic cell separation labelling the CD133 receptor. The growth patterns of isolated CSCs (CD133 positive) in comparison to non-stem cells (CD133 negative) were analysed using real-time cell impedance assays (RTCA). Thereafter, adhesion, invasion and migration assays were performed with the application of small molecule inhibitors. The expression levels of CD133 and SHH were evaluated using confocal microscopy following treatment with cyclopamine.

Results and Discussion: Growth of CSCs appeared to be slower than non-CSCs. Adhesion, invasion and cell migration were inhibited when CSCs were pharmacologically treated either with cyclopamine or SANT-2 (a synthetic analogue of cyclopamine), small molecule inhibitors of the HH pathway. Using confocal microscopy the cell surface expression of Sonic Hedgehog (SHH) was significantly decreased following treatment with cyclopamine, while the expression of CD133 remained unaffected.

Conclusion: Considering these in vitro results, small molecule inhibitors targeting the SHH pathway appear to be promising therapeutic tools for the treatment of metastatic colon CSCs.