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

Aberrant activation of the hedgehog signaling pathway positively correlates with progression, invasion and metastasis of several cancers, including breast cancer. Although numerous inhibitors of the Hedgehog 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 Hedgehog signaling pathway in breast cancer cell lines and tumor-bearing BALB/c mice, using a specific 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 Glioma-associated oncogene homolog 1 (GLI1) transcription factor in a time-dependent manner. We have also shown that elevated level of PKA reduced the level of phosphorylated glycogen synthase kinase 3{beta} (GSK3{beta}), which is known to augment PKA-mediated phosphorylation of GLI2FL and GLI3FL. Rolipram also significantly altered the wound healing capability as well as key markers of wound healing in MCF-7 and MDA-MB-231 cells. Moreover, rolipram caused significant reduction in tumor weight and volume in tumor-bearing mice model. The histological analysis revealed significant reduction in the number of multi-nucleated cells in tumor tissue and substantially reduced levels of cellular infiltration in the lung of rolipram treated tumor-bearing mice. Rolipram also reduced the levels of GLI1 in tumor-bearing mice by enhancing the PKA levels. Therefore, it may be concluded that rolipram can be used as an effective inhibitor of Hedgehog signaling pathway downstream of Smo to control breast cancer progression and metastasis in both hormone-responsive and triple negative breast cancers.