PDE4 inhibitor rolipram represses hedgehog signaling via ubiquitin-mediated proteolysis of GLI transcription factors to regress breast cancer.

Abstract

Aberrant activation of the hedgehog (Hh) signaling pathway positively correlates with progression, invasion and metastasis of several cancers, including breast cancer. Although numerous inhibitors of the Hh signaling pathway are available, several oncogenic mutations of key components of the pathway, including Smoothened (Smo), have limited their capability to be developed as putative anti-cancer drugs. In this study, we have modulated the Hh signaling pathway in breast cancer using a specific FDA-approved phosphodiesterase 4 (PDE4) inhibitor rolipram. The results indicated that increased levels of cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA), due to the treatment with rolipram on MCF-7 and MDA-MB-231 cells, induced PKA-mediated ubiquitination of glioma-associated oncogene homolog 2 full length (GLI2FL) and GLI3FL, leading to their transformation to respective repressor forms. This in turn reduced the level of GLI1 transcription factor in a time-dependent manner. We have also shown that elevated levels of PKA reduced the level of phosphorylated glycogen synthase kinase 3β (GSK3β), which is known to augment PKA-mediated ubiquitination of GLI2FL and GLI3FL. Rolipram treatment also impaired wound healing and migration in both cell lines and significantly reduced tumor weight and volume in tumor-bearing mice. Histological analysis showed a reduction in multi-nucleated cells and cellular infiltration in the lungs of rolipram-treated mice. Moreover, rolipram decreased GLI1 levels in tumors by enhancing cAMP-PKA signaling. These findings suggest that rolipram effectively inhibits the Hh pathway downstream of Smo, offering potential as a therapeutic strategy for controlling breast cancer progression and metastasis, including both hormone-responsive and triple-negative subtypes.