Roles Of Autocrine Cholinergic Signaling In Prostate Development And Cancer Progression

Prostate cancer is the second leading cause of death in male in developed countries.In our country,the incidence of prostate cancer seems to rise duing to the increased life qualities and westernized lifestyles.Androgen deprivation therapy is a common therapy used in prostate cancer treatment.Initially,the prostate cancer will regress after androgen deprivation therapy.However,after a short period of time,the remaining cancer cells will recur and become resistant to androgen deprivation therapy.In addition,during prostate cancer progression,cancer cells may migrate to other parts of the body,most commonly to bone,liver and lymph nodes.Metastasis and castration resistance are the two major challenges in prostate cancer treatment.However,effective treatments targeting prostate cancer metastasis and castration resistance are currently unavailable.In prostate cancer initiation and progression,cancer cells usually hijack the same signaling in prostate development.Our previous studies showed that Wnt signaling,Notch signaling and Sonic hedgehog signaling can regulates both prostate postnatal development and prostate cancer proliferation.However,the signaling that regulates prostate development and prostate cancer progression are largely unknown.In this study,we found that prostate epithelial cells produce both choline acetyltransferase?ChAT? and secrete autocrine acetylcholine.We studied the roles of autocrine cholinergic signaling?ACS? in prostate development and cancer progression.We hope that our studies will provide potential targets for prostate cancer treatment.To study the roles of ACS in prostate development,we performed organotypic cultures of developing ventral prostates?VPs?.We found that carbachol,an acetylcholine analogue,promoted prostatic branching morphogenesis through stimulating the proliferation and inhibiting the differentiation of epithelial progenitor cells.On the contrary,darifenacin,a selective antagonist for CHRM3,inhibited the proliferation of epithelial progenitor cells and induced their differentiation to luminal cells.In lineage tracing assay and renal capsule recombination assay,we also found that ACS regulated the differentiation of prostate epithelial progenitor cells.In addition,we proved that these effects were achieved through Ca²⁺/calmodulin signaling.Calmodulin?CaM? inhibitor W-7could reverse the enhanced branching morphogenesis and increased epithelial progenitor cell proliferation induced by carbachol.Furthermore,we found that activation of muscarinic receptors promoted phosphorylation of Akt both at Ser473 and Thr308.Inhibition of CaM W-7 also effectively reduce carbachol-induced phosphorylation of Akt.Notably,we found that CHMR3 was up-regualted in a large subset of BPH tiusses compared to normal human prostate tissues.Activation of CHRM3 promoted the proliferation of BPH cells.Taken together,our study for the first time demonstrates the roles of autocrine cholinergic signaling in prostate development and BPH cell proliferation.In prostate cancer,we found that CHRM3 was up-regulated in clinical prostate cancer samples.Up-regulated CHRM3 could be activated by endogenous acetylcholine and promoted cancer cell proliferation and migration.On the contrary,blockade of CHRM3 by shRNA or darifenacin inhibited prostate cancer growth and migration.Notably,we found that CHRM3 was up-regulated in matched androgen dependent and castration resistant prostate cancer cells.Over-expression of CHRM3 in androgen sensitive PC3-AR⁺cells caused the castration resistant growth in castrated nude mice.Blockade of CHRM3 via shRNA or darifenacin markedly reduced the established castration resistant ability of PC3 cells.Furthermore,we proved that autocrine cholinergic signaling caused CaM/CaMKK-mediated phosphorylation of Akt.These findings suggest that blockade of CHRM3 may represent a novel adjuvant therapy in prostate cancer treatment,especially in castration resistant prostate cancer treatment.