| Testes-specific protease 50(TSP50)is a novelly identified oncogene firstly found in breast cancer samples and has the activities of threonine proteases.TSP50 is rarely expressed in human normal tissues except testes,but it is highly expressed in various human tumor tissues.Further research shows TSP50 promotes cell proliferation,invasion as well as metastasis and plays an important role in tumorigenesis.Our previous study revealed that TSP50 could promote tumorigenesis partially by activating the NF-kappa B(NF-κB)signaling pathway.In addition,we found that CAGA-luc reporter activity was greatly suppressed by TSP50.Activin is a member of TGFβ superfamily which regulates many cellular events,including inhibiting cell proliferation,so whether TSP50 could promote tumorigenesis by inhibiting activin signal is still unclear.In order to clarify the mechanisms of TSP50 inhibiting activin signal pathway and the correlation between activin signal and TSP50 induced tumorigenesis,we conducted the following research.1.The effect of TSP50 on activin signalActivin signal transmits through the following steps.Activin combines with and phosphorylates receptor type II(ActRII).Phosphorylated ActRII recruits and phosphorylates receptor type I(ActRI).Smad2 and Smad3 are phosphorylated by activated ActRI.Phosphorylated Smad2 and Smad3(p-Smad2/3)enter into nucleus,combine with promoters of the target genes and finally regulate the target genes expression.In order to further confirm our previous results,we first detected the effects of TSP50 on activin signal by overexpression and knockdown of TSP50.(1)The effect of exogenous TSP50 expression on Smad2/3 nuclear translocation in normal cellsIn order to study whether TSP50 has an inhibitory effect on activin signal,we transfected exogenous TSP50 in various normal cells and tested Smad2/3 protein levels in nucleus by western blot.We found that Smad2/3 nuclear translocation was reduced by exogenous TSP50 expression in normal human mammary epithelial(MCF-10A)cells and human normal liver(L02)cells.Moreover,the effects were much more obvious when these cells were stimulated with activin A.These results suggest that exogenous TSP50 can reduce Smad2/3 nuclear translocation in normal cells.(2)The effect of knockdown endogenous TSP50 expression on Smad2/3 nuclear translocation in cancer cellsIn order to further confirm the above results,knockdown endogenous TSP50 expression in human hepatoma HepG2 and three human breast cancer cells ER7530,BT474 and MDA-MB-231 by TSP50 shRNA resulted in accumulated Smad2/3 in nuclei.In summary,these data show that TSP50 inhibits activin signaling pathway by suppressing Smad2/3 nuclear translocation without cell specificity.(3)The effect of TSP50 proteinase activity on Smad2/3 nuclear translocationTSP50 is a novel threonine protease,and its proteinase activity is crucial to TSP50 induced proliferation,invasion,metastasis as well as tumorigenesis.The Thr310 in the catalytic triad is required for its protease activity.In order to study whether TSP50 protease activity plays an important role in TSP50 inhibiting activin signal,we inactivated TSP50 protease activity by mutating Thr to Ala at 310 site of TSP50.We transfected mutant TSP50 T310 A in HEK293 T cells and found that there was no significant inhibitory effect on the expression level of p-smad2/3 and the entry of Smad2/3.This result proves that the threonine protease activity of TSP50 plays an important role in TSP50 inhibiting activin signal.2.The mechanisms of TSP50 inhibiting activin signal(1)The interaction between TSP50 and activin signal elementsIn our previous results,we have confirmed that TSP50 could inhibit activin signal,suggesting that TSP50 might directly or indirectly interact with activin signal elements and inhibit activin signal transduction.Since TSP50 is membrane protein,we consider that TSP50 may interact with membrane receptor of activin signal.In order to determine which receptor combines with TSP50,we transfected TSP50 with myc-ActRIIA or myc-ActRIB in HEK293 T cells.Coimmunoprecipitation assays showed that TSP50 coprecipitated with myc-ActRIIA,but not myc-ActRIB.In order to further clarify the combining site of ActRIIA with TSP50,we transfected TSP50 with extracellular region or intracellular region of ActRIIA in HEK293 T cells.The results detected by mammalian two-hybrid system showed that TSP50 interacted with extracellular region of ActRIIA.(2)The effect of TSP50 on activin receptor phosphorylationThe phosphorylation of type I and type II receptors is required for activin signaling transduction.We then demonstrated whether the combination of TSP50 with receptor II would affect the phosphorylation of the receptor II and thus inhibit the activin signal.In order to verify our hypothesis,we transfected TSP50 with myc-ActRIIA or myc-ActRIB in HEK293 T cells,extracted protein,precipitated with anti-myc antibody and detected the phosphorylation levels of ActRIIA and ActRIB using p-Ser/Thr antibody.Our observations suggested that TSP50 obviously inhibited ActRIIA and ActRIB phosphorylation.Combined with previous results,we speculated that TSP50 could combine with ActRIIA and inhibit ActRIIA and ActRIB phosphorylation.3.The effect of activin signal in TSP50 induced cell proliferation(1)The construction and identification of constitutively activated mutant of ActRIBFrom previous research,we found that TSP50 could inhibit phosphorylation of ActRIIA and ActRIB sequentially and finally inhibit activin signal.So,whether are there some correlations between TSP50-induced inhibition of activin signal and TSP50-induced tumorigenesis? ActRIB activation could be mimicked by mutation of Thr-206 to aspartic acid,we next mutated amino acid T206 to constitutively activate ActRIB.Then we transfected TSP50 with ActRIBT206 D in MCF-10 A cells and detected the expression of Smad2/3 in nucleus.It was found that ActRIBT206 D could significantly reverse the reduction of smad2/3 nuclear translocation caused by TSP50.These results suggest that we successfully construct constitutively activated mutant ActRIBT206 D,and it can be used in subsequent research.(2)The effect of activation of activin signal on TSP50 induced cell proliferationTo prove whether the inhibitory effects of TSP50 on activin signal plays a key role in TSP50 induced tumor cell proliferation,we transfected TSP50 with ActRIBT206 D in MCF-10 A cells and detected the index of cell proliferation by MTT assay,BrdU incorporation assay and Ki67 staining analysis.The data showed that overexpression TSP50 could obviously enhance the vitality of cells and promote DNA synthesis and proliferation related antigen Ki67 expression.But,overexpression ActRIBT206 D could significantly neutralize the effects of TSP50 on promoting cell proliferation.These above results show that the effect of TSP50 on promoting cell proliferation is closely related to its inhibition of activin signal.(3)The effect of activation of activin signal on TSP50-induced tumorigenesisIt has been shown that TSP50 can induce cells anchorage-independent growth and cause nude mice tumorigenesis in vivo.Whether would the activated activin signal by overexpression ActRIBT206 D reverse the cloning formation in soft agar and tumorigenesis in immune-deficient mouse induced by TSP50? We transfected TSP50 with ActRIBT206 D in MCF-10 A cells and detected the effect of ActRIBT206 D on TSP50-induced tumorigenesis.The results demonstrated that ActRIBT206 D could also reverse the TSP50-induced cloning formation and tumorigenesis.Taken together,our results suggest that the activin signal is required for TSP50-induced cell proliferation,and TSP50 promotes tumorigenesis at least partially through inhibiting activin signaling pathway.4.Molecular mechanisms involved in TSP50 promoting cell proliferation via inhibiting activin signalingThe target genes of activin signal,including p21 and p27,are important cell cycle inhibitors,which play a key role in cell proliferation.To investigate the mechanisms of TSP50-promoted cell proliferation via inhibiting activin signal,we discussed the effect of the target gene of activin signal on TSP50-induced cell proliferation.We transfected TSP50 with ActRIBT206 D in MCF-10 A cells and L02 cells and detected the expression levels of p21 and p27 by western blot.The results suggested that TSP50 overexpression decreased p27 protein levels,but had almost no effect on p21 protein levels in both MCF-10 A cells and L02 cells.As is expected,the TSP50-induced reduction of p27 protein levels were also reserved by ActRIBT206 D in the two cells.Additionally,silencing of endogenous TSP50 expression in ER7530 cells led to increased p27 expression,however,this had no obvious effect on p21 levels.These results indicate that TSP50 combines with ActRIIA,suppresses activin signal,inhibits p27 expression and finally promotes cell proliferation and tumorigenesis.5.The expression of TSP50,p-Smad2/3 and p27 in human breast cancer,and the effect of negative relationship between TSP50 and p-Smad2/3 or p27 on diagnosis and prognosis for human breast cancerBecause activin signal is decisive in TSP50-induced cell proliferation in vitro and in vivo,we next investigated whether TSP50 was correlated with the levels of activin signal elements,and whether the correlation between TSP50 and activin signal elements could be regarded as an important diagnostic and prognostic indicators in breast cancer specimens.We detected the expression of TSP50,p-smad2/3 and p27 by immunohistochemistry in the 71 breast cancer specimens.We found that the p-Smad2/3 and p27 protein levels markedly decreased with the increase of TSP50 expression levels.Especially,80%(8/10)and 60%(6/10)of samples that highly expressed TSP50 expressed little p-Smad2/3 and p27 respectively.Moreover,the negative relationship between TSP50 and p-Smad2/3 or p27 was significantly associated with the tumor size,tumor grade and expression levels of some prognostic indicators(ER,PR and her-2).The data suggested that the patients expressing high TSP50 and low p-Samd2/3 or p27 showed higher tumor grade,and easier to show ER and PR positive,and HER-2 negative than the patients expressing low TSP50 and high p-Samd2/3 or p27.In summary,this study systematically discussed the molecular mechanisms of TSP50 inhibiting activin signal and the correlations between activin signal and TSP50-induced tumorigenesis.We found that TSP50 could combine with ActRIIA,inhibit phosphorylation of ActRIIA,ActRIB and Smad2/3 sequentially,repress the accumulation of Smad2/3 in nucleus,reduce the expression of activin signal target gene p27,affect cell cycle and finally promote cell proliferation and tumorigenesis.Furthermore,the inverse correlation between TSP50 and activin signaling was associated with clinicopathological feature,and our results might provide a strategy for future diagnosis and prognosis of human breast cancer. |
PDF Full Text Request