SIGNALING CROSSTALKS IN CANCERS: COOPERATIONS AMONG RAS, HEDGEHOG, cAMP AND GLUCOCORTICOID

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Increased intracellular cAMP can either inhibit or promote apoptosis, depending upon the specific cellular context ⁵⁰. The determinants for cAMP-mediated cell cycle arrest and cell death are poorly understood. Previous studies on CEM cell lines have documented that cAMP elevating agents such as forskolin synergistically promote Dex induced cell death in both GC-sensitive and resistant CEM clones ⁵², thus implicating the involvement of cAMP signaling in the GC- induced apoptotic process. In the current studies, we demonstrate that between the two intracellular cAMP receptors, Epac and PKA, PKA is responsible for the synergistic killing effects observed in CEM cells. This is consistent with a previous report that cAMP exerts its growth arrest effect through PKA instead of Epac pathway in Jurkat T cells ²⁵⁹. Activation of Epac, on the other hand, weakly antagonizes the apoptotic effect of Dex, consistent with our earlier observation that Epac and PKA can mediate the opposing cellular effects of cAMP ²⁶⁰. 

In agreement with the important role that PKA plays in cAMP-mediated synergism with GC, the cellular activity of PKA is significantly higher in GC-sensitive CEM cells. This increased PKA activity is further associated with a decreased Hh activity in GC-sensitive C7-14 cells. We also find that forskolin suppresses Hh pathway activity in both GC-resistant and sensitive CEM cells. Moreover, while Dex alone does not significant suppress Hh activity, the inhibitory effect of forskolin on Hh activity is synergistically potentiated by Dex. Therefore, our studies for the first time suggest that inhibition of Hh pathway activity by PKA may represent one of the mechanisms that cAMP and GC converge to induce cell apoptosis in CEM cells.

Hh signaling regulates cell proliferation, differentiation and survival in some adult tissues in addition to its important roles during development ⁵⁸. Dysregulation of the Hh pathway has been implicated in many human cancers ^48,261. Our findings that the Hh pathway is activated in acute lymphoblastic leukemia cells broadens the growing list of Hh activated cancers. While the mechanism underlying Hh-mediated tumorigenesis is poorly understood, several studies indicate a role for Hh as a regulator of the cell cycle machinery ²⁵³. These studies are in agreement with our observations that inhibition of Hh pathway by a SMO inhibitor KAAD-cyclopamine leads to cell cycle arrest in G1/S, while a structurally-related analog, tomatidine, shows no effect on the cell cycle progression. Hh pathway blockade by pharmacological agents or small interference RNA (siRNA) induce cell growth arrest and apoptosis in several solid tumors, as well as inhibit tumor growth in mouse xenograft models ^141,237,262. Again, these studies are consistent with our findings that KAAD-cyclopamine induces apoptosis in CEM cells.

PKA is negative regulator of Hh signaling. In mammalian system, Hh-activated transcription factors, Gli2 and Gli3, are sequentially phosphorylated by PKA, glycogen synthase kinase 3 and casein kinase I, before being targeted for proteolytic processing to generate a  repressor form  or completely degraded by the 26S proteosome complex ²⁶³. In addition to affecting Gli protein stability, phosphorylation of Gli1 by PKA also prevents Gli1 translocating from cytoplasm to nucleus in COS7 cells ⁵⁷. Consistent with these observations, our study indicates that PKA down-regulates Hh pathway activity and PKA/Hh activities correlate with GC-sensitivity in acute lymphoblastic leukemia cell lines. Our findings that increased PKA activity suppresses basal Hh activity in GC-sensitive cells provide a molecular explanation for the synergistic killing effects between GC and cAMP. Most importantly, this relationship between PKA/Hh activities and GC-sensitivity is confirmed in the GC-sensitive revertant C1-8 cell line, which also contains a higher level of PKA activity, lower Hh pathway activity, and increased sensitivity to Hh inhibition.

Synergy between cAMP/PKA and GCs in promoting apoptosis perhaps involves cooperative effects on gene transcription by GRs and the PKA pathway ⁵². Transcriptianally activation of BIM expression has been implicated as a common target for both PKA and GCs ⁵³. It has been shown that GC treatment of sensitived clones, on GC and forskolin treatment of a resistant clone, lead to increased BIM expression ^244,245. Our results that forskolin and Dex synergistically suppress Hh pathway activity and promote apoptosis in CEM cells provide another important mechanism towards understanding the synergy between PKA and GCs.

Overall, our results suggest that suppressing Hh pathway is an important part of the mechanism of synergistic cell killing of lymphoid cells by GCs and PKA. Overactivation of Hh signaling in GC resistant CEM cells may be one of the mechanisms leading to GC resistance. The Hh pathway thus appears to be a molecular target of cAMP/PKA signaling and in part determines GC sensitivity in ALL. This finding is very important from the clinical viewpoint, as it suggests that Hh pathway inhibitors may be used to effectively treat GC-resistant leukemia.