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