A large and diverse array of cancer types show evidence of overactive HH signalling [
11]. This has triggered an intensive search for agents that selectively inhibit this pathway [
20]. As a result, several molecules have been discovered or designed that directly interact and inhibit the activity of SMO, some of which are in clinical trials [
20]. Despite the promise of this approach, two potential problems are that 1) some tumors acquire mutations in SMO that render them relatively resistant to SMO antagonists [
45], and 2) SMO is critically involved in the growth of bones and other tissues [
11,
46], and appears to regulate stem cell biology [
9] and [
47]. Thus, there is a need for molecules that act downstream of SMO in the HH signalling pathway and that inhibit HH signalling specifically in tumor cells. PKA is thought to act in the HH pathway downstream of SMO, phosphorylating and triggering proteosome-mediated degradation of Gli2 and Gli3 [
48,
50]. Thus PKA activators might be expected to block HH signalling even in the context of drug-resistant SMO mutations. However, given that PKA is used in so many biological processes, it is unlikely that general activation of PKA will be useful as a treatment strategy. An alternative strategy would be to activate PKA specifically in tumor cells. This could potentially be achieved by targeting appropriate heterotrimeric G protein-coupled receptors expressed on the cells. The data reported here suggest that a potentially-effective strategy to treat MB tumors exhibiting overactive HH signaling would be to activate PACAP receptors. We investigated this possibility by using genetically engineered mice that spontaneously develop MB, and a tissue culture system that preserves a significant level of constitutive HH activity in tumor cells. We found that PACAP inhibited proliferation and the expression of the HH target gene
gli1 in these cells. Moreover, the reduction of
gli1 gene expression by PACAP was reversed by PKA inhibition, and was mimicked by stimulation of the cAMP/PKA pathway by forskolin. Overall, the data suggest that PACAP, via activation of PKA, inhibits HH signalling and HH-driven proliferation in MB cells. However, our findings do not rule out alternative mechanisms of PACAP/PKA action in these assays. For example, despite its action on
gli1 in MB tumorspheres, the ability of PACAP to inhibit proliferation might be unrelated to its effects in HH pathway activity. In this respect, PACAP (and PKA activation in general) is known on inhibit the proliferation of many types of cells [
51,
53], some of which might not exhibit HH pathway activity. In any case, the findings reported here suggest that PACAP or PAC1 agonists could be useful as alternatives to, or in conjunction with, SMO antagonists, for the treatment of MB and other HH dependent tumors.