prostate cancer ( pca ) , the most frequently diagnosed malignancy , has become the second leading cause of cancer - related deaths among men in western countries [ 1 , 2 ] .
endocrine therapies which aimed at inhibiting the androgen receptor ( ar ) function was the mainstay of treatment for advanced prostate cancer based on that the androgen signaling will promote the proliferation of prostate cancer cell . unfortunately , most of treated patients progressed toward castration - resistant prostate cancer ( crpc ) from castration - dependent prostate cancer . and
crpc characterized by aggressive growth and ability to colonize distal organs , which made crpc still incurable and the median survival time for patients with crpc was only 12 months .
the status of ar was highly predictive of prostate cancer patients that will benefit from endocrine therapy but was not correlated with a better clinical outcome [ 4 , 5 ] .
prostate - specific antigen ( psa ) is a protein produced by the prostate gland cells .
although many published studies have assessed the performance of candidate biomarkers in predicting time to relapse of prostate cancer following radical prostatectomy [ 6 , 7 ] , no molecular markers suitable for routine clinical practice that can identify those prostate cancer patients with a high risk of early clinical progression or prostate cancer - specific mortality have been found .
g - proteins are composed of  ,  , and  subunits and  subunit are classified into 4 families : gs , gi / o , gq/11 , and g12/13 .
each of them has multiple members with different expression specificity [ 8 , 9 ] .
although gs is the most extensively characterized and clinically relevant , literature is not unanimous on the role of gs in different types of cancers . in lung cancer ,
choi et al . found that gs could augment cisplatin - induced apoptosis of lung cancer cells through upregulating bak expression by increasing transcription and by decreasing the rate of protein degradation   and the efficacy of radiotherapy of lung cancer may be improved by modulating gs signaling pathway .
but in cervical cancer or intrahepatic cholangiocarcinoma ( icc ) , the situation was just opposite .
cho et al . found gs inhibited cisplatin - induced apoptosis by increasing transcription of x - linked inhibitor of apoptosis protein ( xiap ) and by decreasing degradation of xiap protein in hela cervical cancer cells . in icc , schmitz et al . also found a significant association of both unfavorable disease - specific overall survival and recurrence - free survival with the homozygous tt genotype of the gnas1 gene which encoded gs protein [ 13 , 14 ]
however , they also reported that t393c polymorphism in the gene gnas1 was significantly associated with favorable clinical outcome of patients suffering from bladder cancer , chronic lymphocytic leukemia , and renal cell carcinoma .
there were studies reported no association was found between gnas t393 t genotype and prostate cancer [ 17 , 18 ] . but liu et al . identified that membrane caveolae - associated gas was involved in androgen receptor ( ar ) transactivation by modulating the activities of different pi3k isoforms .
more importantly , it had been reported that the expression of gs and gi decreased 30% to 40% after neoplastic transformation . and
the levels of gs and gi subunits correlated inversely with serum prostate specific antigen in patients with prostate cancer , which indicated an important regulatory role of gs and gi for cell proliferation and neoplastic transformation in human prostate cancer and they may have prognostic value . therefore , more in - depth investigations are necessary to address this controversy and identify the role of gs in prostate cancer .
thus , we assessed the potential of gs as a prognostic marker by determining the level of gs protein expression in a series of 347 postradical prostatectomy prostate cancer tissue microarrays ( tma ) which include 56 metastatic pcas and 291 localized pcas and 67 benign prostatic hyperplasia ( bph ) as controls using immunohistochemistry ( ihc ) . in the present study
, we found that expression of gs protein was decreased in high grade and metastatic pcas . and low gs protein levels were strongly associated with adverse clinicopathologic features and poor clinical outcomes in metastatic and localized pca patients .
multivariate cox regression analysis showed that low expression of gs was an independent predictor of prostate cancer recurrence and cancer - specific death in metastatic and localized pca . to the best of our knowledge ,
this is the first study to identify the independent predictive role of gs in patient with prostate cancer .
in order to study gs expression in prostate cancer by immunohistochemistry , a total of 347 formalin - fixed , paraffin - embedded prostate tissues between 1994 and 1997 were retrieved from the archives of the first affiliated hospital , college of medicine , and xi'an jiao tong university , and a tissue microarray ( tma ) was constructed .
in addition , 67 benign prostatic hyperplasia ( bph ) samples were collected as control .
this research project was approved by the ethical committee of the xi'an jiao tong university , and all the patients had been given their fully written informed consent .
data were collected on patients with disease baseline and clinicopathologic characteristics as well as 2 treatment outcomes : time to progression and prostate cancer - specific mortality ( pcsm ) .
prostate cancers were graded based on the gleason system by 2 independent pathologists at the first affiliated hospital , college of medicine , and xi'an jiao tong university in a blind and consecutive manner to ensure adequate diagnosis and grade .
the tnm staging system was used to describe the extent of prostate cancer in patients ( based on the ajcc cancer staging manual , seventh edition , 2010 , springer , new york , inc . ) .
briefly , slides were deparaffinized in xylene and rehydrated in a graded alcohol series before endogenous peroxidase activity was blocked with 3% h2o2 in methanol .
after nonspecific protein binding was blocked , the primary antibody diluted into recommended concentration for gs , which was purchased from abcam ( ab58810 ) , was applied overnight in a humidity chamber at 4c .
biotinylated secondary antibody was applied for 30  min at room temperature after washing with pbs for 3 times .
negative control was conducted by replacing the primary antibody with preimmune rabbit serum . to evaluate gs expression
, we used the immunoreactive score ( irs ) as previously implemented by tischler et al .
the intensity of staining was arbitrarily graded as absent ( 0 ) , weak ( 1 + ) , moderate ( 2 + ) , and strong ( 3 + ) . the percentage of stained cells was use d to quantify the react ion as negative ( 0% of positive cells ) , 1 + ( < 10% positive cells ) ; 2 + ( 1050% of positive cells ) ; 3 + ( 5180% of positive cells ) ; 4 + ( > 80% of positive cells ) . the final value of the analysis of each tissue sample was then expressed as an absolute value through the obtained score by multiplying the two individual scores ( i.e. , intensity of staining score times the percentage of stained cells score ) then generates a final score ranging from  ( no expression ) to + ( weak expression ) , + + ( moderate expression ) , or + + + ( strong expression ) . and we identified  and + as negative for gs expression and + + and + + + as positive gs expression .
examples of scoring according to staining intensity and the percentage of stained cells are shown in figure 1 .
spss version 13.0 ( spss , chicago , il , usa ) was used for statistical analyses .
psa progression - free and overall survival curves were constructed by the kaplan - meier method and compared using the log - rank test . to evaluate the role of prognostic variables , a series of cox proportional hazards models
since psa was a continuous estimate , with the median psa level for the entire cohort of patients ( n = 347 ) as 34.9  ng / ml , we divided the cohort into those with psa levels 35  ng / ml and > 35  ng / ml . the following parameters were included : psa levels ( 35  ng / ml , > 35  ng / ml ) ; extraprostatic extension ( yes , no ) ; involvement of surgical margins ( no , yes ) ; involvement of seminal vesicles ( no , yes ) ; involvement of pelvic nodes ( n0 , n+ ) ; gleason scores ( 26 , 7 , 810 ) .
145 patients ( 41.8% ) presented gleason score of 26 , 127 ( 36.6% ) patients presented gleason score of 7 , and the remaining 75 cases ( 21.6% ) presented gleason score between 8 and 10 .
49 patients ( 11.5% ) presented tnm stage i ; 125 ( 36.0% ) patients presented stage ii ; 117 ( 33.7% ) presented stage iii ; and 56 ( 16.1% ) patients presented tnm stage iv .
psa progression was observed in 229 ( 66.0% ) patients at a median interval of 123.5 month ( range 7167 ) .
other clinicopathological features are summarized in table 2 . moreover , prostate cancer patients who had higher gleason scores ( p < 0.001 and p < 0.001 , resp . ) , higher tnm stages ( p < 0.001 and p < 0.001 , resp . ) , higher preoperative psa level ( p < 0.001 and p < 0.001 , resp . ) , positive surgical margin ( p = 0.009 and p < 0.001 , resp . ) , angiolymphatic invasion ( p = 0.004 and p = 0.032 , resp . ) , extraprostatic extension ( p = 0.031 and p < 0.001 , resp . ) , and seminal vesicle invasion ( p = 0.046 and p = 0.007 , resp . )
present shorter overall survival and psa progression - free survival ( tables 5 and 6 ) .
psa progression and overall survival time correlated with tnm stage , gleason score , extraprostatic extension , positive surgical margins , and seminal vesicle invasion demonstrate the representability of study group .
the number of patients with positive lymph node involvement ( n = 34 ) was too small to find any significant correlation with psa progression - free survival and overall survival . to determine the prevalence and clinical significance of gs in prostate cancer tissues
, we determined the expression of gs protein by immunohistochemistry in a retrospective cohort of 347 tumor tissue samples from prostate cancer patients and 67 samples from patients who were diagnosed with benign prostatic hyperplasia ( bph ) after tumor resection . among the 347 patients , 114 patients
had not expression of gs (  ) ; 73 patients were weak expression ( + ) ; 86 patients were moderate expression ( + + ) , and 74 patients were strong expression ( + + + ) ( as shown in figure 1 ) .
thus , as we described in the methods section , there were 160 ( 46.1% ) samples positive for gs expression and 187 ( 53.9% ) samples negative for gs expression in our pca cohort .
we also found that positive ratio of gs expression was downregulated in metastatic pca compared to localized pca and bph ( p = 0.012 and p < 0.001 , respectively , as shown in table 1 ) . in patients with bph
, there were 42 ( 62.7% ) samples positive for gs expression and 25 ( 37.3% ) samples negative for gs expression . in the patients with localized pca
, there were 151 ( 51.2% ) samples positive for gs expression and 140 ( 48.1% ) samples negative for gs expression .
whereas in the patients with metastatic pca , there were 9 ( 16.1% ) samples positive for gs expression and 47 ( 83.9% ) samples negative for gs expression .
ihc staining for gs was sharp and reliable , no background or nonspecific staining was observed .
then , we further evaluated the relationship between gs expression and clinical features of prostate cancer patients by pearson chi - square test or fisher 's exact test .
we found that the positive ratio of gs expression was decreased as the level of gleason score or preoperative psa increased .
these result showed that there was inverse correlations between gs expression and preoperative psa and gleason score and tnm stage at diagnosis ( p = 0.030 , p < 0.001 , and p < 0.001 , respectively , table 2 ) .
but we found no specific correlation between gs expression and the rest of pathological parameters ( age ; angiolymphatic invasion ; extraprostatic extension ; positive margin ; seminal vesicle invasion ; positive lymph node ) that we evaluate in the present analysis . in the localized pca specimens , the expression of gs
was correlated with preoperative psa level , gleason score , and tnm stage ( p = 0.028 , p = 0.016 , and p = 0.011 , respectively , table 3 ) . however , in metastatic pca specimens , the expression of gs was only associated with preoperative psa level and gleason score much more significantly ( p < 0.001 , and p < 0.001 , resp . , table 4 ) . in our retrospective cohort with 347 patients
, we got the detailed follow - up information of 15 years . at the time of our analysis , 121 patients died and 256 patients progressed .
median time to psa progression for the whole cohort was 123.5 months ( range 7167 months ) , while the median time to death was 123.5 months ( range 3179 months ) .
we found that patients with negative expression of gs had a higher ratio of psa progression than those with positive gs expression ( table 2 ) .
more importantly , negative gs expression was associated with psa progression - free survival and overall survival .
the group of patients with negative expression of gs showed significantly shorter overall survival than patients with positive expression of gs ( p = 0.001 , figure 2(a ) ) .
these patients also showed a trend for shorter psa - free survival time ( p < 0.001 , figure 2(d ) ) .
in localized pca specimens , negative gs expression was also associated with better psa progression - free and overall survival rate ( p < 0.001 , figure 2(c ) ; p < 0.001 , figure 2(f ) ) . in metastatic
pca specimens , a similar trend was found between negative gs expression and psa progression - free / overall survival time ( p = 0.0003 , figure 2(e ) ; p = 0.0146 , figure 2(b ) ) . as expected , at the univariate level , gleason scores , tnm stages , preoperative psa , positive margin , angiolymphatic invasion , extraprostatic extension , and seminal vesicle invasion were associated with psa progression - free and overall survival .
negative expression of gs protein was a prognostic predictor of psa progression - free and overall survival in pca patients at univariate level ( tables 5 and 6 ) .
we further conducted a multivariate cox regression analysis to assess whether gs was a prognostic predictor of survival independent of age , gleason scores , tnm stages , preoperative psa , positive margin , angiolymphatic invasion , extraprostatic extension , seminal vesicle invasion , and positive lymph node .
multivariate analysis showed that negative gs expression was a strong independent predictor of outcome providing survival information ( both psa progression - free or overall survival ) above other independent prognostic features ( tnm stage , gleason score ) , with a hazard ratio of 4.328 and 3.904 and a 95% confidence interval of 1.8768.432 , p < 0.001 ( negative gs group versus positive gs group ) and 1.2785.873 , p < 0.001 ( negative gs group versus positive gs group ) . in our cohorts , psa , positive margin , angiolymphatic invasion , extraprostatic extension , and seminal vesicle invasion
it has been reported that the expression of gs correlated inversely with serum prostate specific antigen in patients with prostate cancer and the expression of gs decreased 30% to 40% after neoplastic transformation .
but there was no study concerning the role of gs protein in the prognosis of prostate cancer patients . in the present study
, we characterized the expression pattern of gs protein in a large number of tissues derived from prostate cancer patients , consisting of localized and metastatic pca , and assessed the utility of gs as a prognostic marker in these patients . in agreement with previous reports
, we confirmed that gs expression was localized in nuclear and cytoplasm in neoplastic cells .
moreover , we found that expression of gs was downregulated in metastatic pca compared to localized pca and bph . and
gs was inversely associated with psa level and gleason scores both in localized and metastatic pca . at the univariate level , gs , gleason scores , tnm stages , preoperative psa level , positive margin , angiolymphatic invasion , extraprostatic extension , and seminal vesicle invasion
but in multivariable cox regression analysis , only high gs expression and gleason scores were independent predictors of both psa progression - free and overall survival .
these findings support the potential clinical utility of incorporating gs into clinical nomograms to help determine the risk of psa progression and death .
the prognostic significance of gs as biomarker for prostate cancer is likely to its biological functions .
gs is a member of gtp - binding protein superfamiliy and could independently regulate a variety of effectors including adenylate cyclases , phospholipase c , and ion channels [ 22 , 23 ] .
however , gs and t393c polymorphism in the gene gnas1 which is encoded by gs plays distinct roles in different cancers .
gs could augment cisplatin - induced apoptosis of lung cancer cells , but it inhibited cisplatin - induced apoptosis in cervical cancer or intrahepatic cholangiocarcinoma ( icc ) .
t393c polymorphism in the gene gnas1 was significantly associated with favorable clinical outcome of patients suffering from bladder cancer , chronic lymphocytic leukemia , and renal cell carcinoma , and significantly associated with unfavorable clinical outcome of patients suffering from icc and breast cancer [ 13 , 14 ] . in prostate cancer
, previous study reported that low expression level of gs was found in t2 stage pca compared to high levels in normal controls .
more importantly , the expression of gs was found downregulated in hormone refractory c4 - 2b and pc3 cell lines compared to hormone sensitive lncap and rwpe-1 cell lines .
all these studies indicating the functionality and expression of gs are selectively modified in human prostate adenocarcinoma and downregulated gs levels may play an important regulatory role for cell proliferation and neoplastic transformation in prostate cancer .
thus , we did our efforts to investigate and validate whether gs functioned as an efficient prognostic biomarker to predict the outcome of pca patients .
consistent with previous studies , we found that the expression of gs was much lower in metastatic pca than it is in localized pca .
furthermore , the patients who had the low expression of gs tend to have shorter progression - free survival and overall survival time , nonetheless , metastatic or localized pca .
more importantly , multivariable cox regression analysis proved that gs was an independent predictor of prognosis in prostate cancer .
though the answer to the discrepancy of gs function in different cancers was still unclear , and the role of gs in prostate cancer was also in dispute , our results were relatively easy to understand for gs had a close relationship with egfr .
egfr belongs to erbb oncogene family which also includes erbb-2 , 3 , and 4 and is comprehensively expressed in epithelial cells including prostate cancer cells .
egfr are known to regulate cell proliferation , differentiation , angiogenesis , and survival . in prostate cancer ,
it has been reported that egfr was highly expressed in du145 and pc3 cell lines which were hormone - independent human prostate cancer cell lines and responsive to egf stimulation [ 2527 ] .
it was also found that prostate cancer bone metastases express significantly higher level of egfr .
more importantly , egfr expression increased as prostate cancer progressed from an androgen - dependent to an androgen - independent stage .
di lorenzo et al . , found 41% , 76% , and 100% egfr expression in radical prostate ectomy hormone - sensitive and hormone - refractory metastatic patients in a cohort consisting of 76 patients with androgen - dependent and androgen - independent prostate cancer , respectively .
many studies confirmed that overexpression of egfr contributes significantly to the progression of prostate cancer [ 3134 ] .
zheng et al . reported that overexpression of the stimulatory gs promotes ligand - dependent degradation of epidermal growth factor ( egf ) receptors and texas red egf , and knock - down of gs expression by rna interference ( rnai ) delays receptor degradation .
recently , they demonstrated that egf - induced , proliferative signaling occurs from eea1 endosomes and was regulated by the gs through interaction with the signal transducing protein giv ( also known as girdin ) .
when gs or giv was depleted , activated egfr and its adaptors accumulate in eea1 endosomes .
then egfr signaling was prolonged and egfr downregulation was delayed , which made cell proliferation greatly enhanced . basing on
our finding that gs was downregulated in advanced pca and the previous studies concerning the function of egfr in pca , we hypothesise that downexpression of gs inhibit the degradation of egfr , then androgen receptors which are activated by egfr were prolonged and cell proliferation increased , eventually causing tumor progression and hormone - resistant in prostate cancer . that maybe the underlying mechanism account for our results in which the expression of gs was downregulated in metastatic pca and
inversely associated with psa level and gleason scores . but experiments would be desirable to further clarify the relationship between gs and egfr and identify their function in the progression of prostate cancer . however , such functional studies were beyond the scope of this study .
in summary , we discovered that gs is a promising biomarker of prostate cancer patients . to our knowledge , this is the first study to describe the predictive role of gs in prostate cancer .
we found that the expression of gs was downregulated in metastatic prostate cancer compared to localized prostate cancer . and
although our results are promising , gs expression needs to be validated in relationship to outcome in the context carefully controlled clinical trials .
all in all , targeting gs could be a promising therapeutic strategy for enhancing the therapy effect of patients .
 
 background . 
 t393c polymorphism in the gene gnas1 , which encodes the g - protein alpha s subunit ( gs ) of heterotrimeric g protein , is significantly associated with the clinical outcome of patients suffering from several cancers . 
 however , studies on the role and protein expression of gs subunit in prostate cancer were still unavailable . methods . 
 the immunohistochemical staining was used to assess gs expression through tissue microarray procedure of 56 metastatic pcas , 291 localized pcas , and 67 benign hyperplasia ( bph ) . 
 gs expression was semiquantitatively scored and evaluated the correlation with pathologic parameters and biochemical recurrence of prostate - specific antigen ( psa ) . results . 
 gs expression was localized in nuclear and cytoplasm in prostate cancer cells and downregulated in metastatic pca compared to localized pca and bph ( p < 0.001 ) . 
 gs was inversely associated with psa level and gleason scores ; patients with low expression of gs had adverse clincopathological features . 
 in multivariable cox regression analysis , high gs expression and gleason scores were independent predictors of both psa progression - free and overall survival . conclusions . 
 gs down - expression is associated with adverse pathologic features and clinical psa biochemical recurrence of prostate cancer . 
 gs is an independent predictor to help determine the risk of psa progression and death . 
1. Introduction
2. Patients and Methods
3. Results
4. Discussion
5. Conclusion