Epigenetic drugs such as HDAC inhibitors regulate

Epigenetic drugs, such as HDAC inhibitors, regulate gene expression by affecting the activity of histone or DNA modifying enzymes and their associated transcriptional response [141]. BET bromodomain protein inhibition is another epigenetic approach for blocking the Hedgehog pathway at the downstream level. For instance, JQ1 has been developed as a selective inhibitor of BET bromodomains, showing inhibitory ability upon cell viability and proliferation in vitro and in vivo in models of Smo-antagonist resistant medulloblastoma [142,143].
SHh crosstalk with TGF/SMAD signaling Based on the insights gleaned from research over the course of decades, it has become evident that cancer phenethyl and bone microenvironments interact with each other through various signaling molecules [144]. Cancer cells produce angiogenic factors and bone resorbing factors which significantly enhance the proliferation of cancer cells within the bone microenvironment. More excitingly, the bone tissue acts as a repository for growth factors, such as bone morphogenetic proteins (BMPs) and transforming growth factor (TGF)-β. BMP-4 was noted to upregulate the SHh mRNA in prostate cancer CWR22 cells, an event that induces osteoblastic lesions when injected into tibia of immunodeficient animal models. BMP-4, BMP-6 and BMP-9 upregulated SHh mRNA expression in LNCaP cells in a dose dependent manner, though the effects of BMP-4 and BMP-6 were more pronounced that the ones associated with BMP-9. Mechanistically it was shown that BMP-4 increases SHh production through SMAD4 modulation in prostate cancer cells [144]. As expected, BPM-4 mediated increase in SHh was notably reduced in SMAD4 silenced LNCaP cells. BMPs were reported as being involved in osteoblastic differentiation of stromal cells, and treatment of stromal MC3T3-E1 cells with SHh considerably increased the BMP-responsive reporter activity induced by BMP-4. SHh markedly increased BMP signaling in MC3T3-E1 cells by enhancing the expression levels of BMP signaling modulators. SHh significantly upregulated activin receptor and SMAD1 expression in MC3T3-E1 cells. Furthermore, BMP-4 mediated phosphorylation of the C-terminal region of SMAD1 increased notably in MC3T3-E1 cells treated with SHh [144]. Yet another important observation was that LNCaP cells produced SHh in response to BMPs. Moreover, BMPs and BMP-induced SHh worked synergistically and facilitated the osteoblastic differentiation of MC3T3-E1 cells. BMP-4 also inhibited the growth of LNCaP in monoculture conditions. However, when MC3T3-E1 cells were co-cultured with LNCaP, BMP-4 mediated growth inhibition was abolished. Surprisingly, BMP-4 promoted LNCaP growth, suggesting that MC3T3-E1 cells supported growth and/or survival of LNCaP cells [144].
SHh crosstalk with PDGFR Treatment of BRAF (V600E) metastatic melanoma with BRAF inhibitors is challenging due to acquired and intrinsic drug resistance. It has previously been convincingly determined that the use of BRAF inhibitors triggers SHh pathway activation with consequences on the upregulation of PDGFRα (Platelet-derived growth factor receptor α) [145]. Due to activation of the SHh pathway in BRAF inhibitor treated melanoma cells, SHh inhibitors were tested. Data clearly suggested that SHh and BRAF inhibitors synergistically inhibit SHh and PDGFRα in melanoma cells. SHh inhibitor (40 mg/kg/day) enhanced the efficacy of vemurafenib to inhibit tumor growth of M21 cells in SCID mice [145]. PDGRFα was noted to be significantly downregulated in sulforaphane administered xenografted mice. Gli1 and Gli2 were also observed to be repressed in mice xenografted with pancreatic cancer stem cells [146].
Notch signaling The notch signaling pathway participates in multiple cell processes including differentiation, proliferation, and survival [147]. Notch genes encode transmembrane receptors which have been shown to be upregulated together with their ligands in many types of cancer [148,149]. A well-established crosstalk between Notch signaling and the Hedgehog pathway has been reported by various authors in breast, multipotent mesodermal, glioblastoma and in prostate cancer cells with acquired resistance to docetaxel [[150], [151], [152], [153]]. It has been suggested that Notch can directly suppresses the Hedgehog pathway via the repressive transcription factor Hes1, by inhibiting Gli1 transcription [153]. In an in vivo animal model of ovarian cancer, Jagged1, a Notch ligand, reduced tumor growth partially through a crosstalk mechanism with the Gli2 mediator [154].