The Function And Related Mechanisms Of MYB In Salivary Glands Adenoid Cystic Carcinoma

Adenoid cystic carcinoma(ACC)is one of the most aggressive malignancies that develop in the salivary gland,accounting for 21%-24%of the salivary gland cancer.With the characteristics of nerve-devoured growth,more than 60%of the patients succumb to the disease as a result of the recurrence and distant metastasis.The patients'5-year survival rate is high,but the 10-years or even longer survival rate is much lower.Because ACCs are very easy to relapse after surgery,it is a much tricky problem that we do not have effective clinical treatment of ACC at present.Salivary gland adenoid cystic carcinoma(SACC)is not sensitive to chemotherapy;accordingly,surgery combined with postoperative radiotherapy still is the main treatment of SACC.Because of unsatisfactory conventional treatment effect,we need to further understand the biological phenotype and molecular genetic characteristics of SACC,trying to provide new viewpoint and treatment approaches.With the development of molecular mechanisms investigations of ACC,the researchers find gene therapy may represent a major breakthrough that is expected to facilitate the generation of new therapies of SACC.Through the study on the genes that play a regulatory role in the development of tumors,we may inhibit the proliferation of tumor cells and induce tumor cell apoptosis in SACC by regulating the oncogene expression.The molecular pathogenesis of ACC is a very complex process in which the translocation between chromosome 6q and 9q((6;9)(q22-23;p23-24))plays a very important role.Dr.Persson's group first reported this translocation can lead to MYB and NFIB gene fusion.This fusion is ACC-specific,accounting for 86%of all ACC causes.Therefore,ACC is different from other tumors molecularly.MYB-NFIB fusion will lead to MYB overexpression and alterations of MYB targeting genes expression that results in tumorigenesis.At present,studys found that about 40%of human SACC cases have MYB-NFIB gene fusion.Genome sequencing also confirmed MYB gene alterations prevalently exist in SACC.At the same time,more than 50%of the human SACC cases without MYB-NFIB fusion are found to contain full length MYB overexpression,which do not occur in other salivary gland tumor types.Moreover,studies have shown that the interaction between MYB and its downstream signaling pathway also plays an important role in the occurrence of SACC.These studies suggest that MYB alterations may play a major role in SACC development.Due to the lack of effective study models,there is no report on the role of MYB overexpression in the development of SACC result from MYB alterations.Accordingly,we hypothesized that MYB overexpression is an important factor in the development of SACC as a proto-oncogene"promoter".In this project,we used the TRE-MYB mice and MMTV-tTA transgenic mice to establish the TRE-MYB-M.tTA transgenic mouse model in which salivary and mammary gland specifically overexpress MYB in order to do the further research on the function of MYB gene in SACC development.This project was divided into the following three parts to study.Part I:Generate the TRE-MYB transgene.Co-transfected cells using TetO-MYB and CMV-rtTA plasmids.The sensitivity of mice and human cells to the Tet-On system was compared by observing the expression level of MYB under doxycycline.The TRE-MYB transgenic mouse model was established on the basis of the Tet-On system.After that,the TRE-MYB mice were crossed with MMTV.tTA mice to obtain a TRE-MYB-M.tTA(+/+)adult mouse model that specifically overexpressing MYB in salivary and mammary glands.The salivary gland tumors were collected from the TRE-MYB-M.tTA mice and identified from histopathological point.Then,the expression levels of p63 and c-KIT were examined.Part II:Examine the expression level of MYB in SACC developed in TRE-MYB-M.tTA mice and the effect on SACC when MYB expression was inhibited by treating with doxycycline.Part III:Examine the expression level of NOTCH-1 in SACC developed in TRE-MYB-M.tTA mice.Part IObjectTo generate TRE-MYB-MtTA(+/+)Transgenic Mouse Model and identify the SACCs developed in this model.DesignWe transfected mouse SCC and human ACC cells with CMV-rtTA and TetO-MYB plasmids and observed the expression levels of MYB under the efficacy of doxycycline.And then,we generated TRE-MYB transgenic mice after the characterization of TRE-MYB transgene was confirmed.The TRE-MYB mice were hybridized with MMTV.tTA mice which received from Dr.Wagner's Laboratory,Nebraska Medical Center in the United States.We selected out the TRE-MYB-M.tTA(+/+)mice from their progeny and fed them continuously.When TRE-MYB-M.tTA mice developed tumor,we collected and diagnosed the tumor from histopathological point.At the same time,we tested the expression of p63 and c-KIT at the protein level.The normal salivary gland tissues were used as blank controls.ResultThe expression of MYB in cells transfected by CMV-rtTA and TetO-MYB plasmids was able to be regulated by doxycycline,and TRE-MYB transgenic mice were successfully established on this base of TRE-MYB transgene.TRE-MYB-M.tTA(+/+)mice were successfully screened by hybridization TRE-MYB and MMTV.tTA mice.There were 9 TRE-MYB-M.tTA mice developed tumors.8 of them developed salivary gland tumors(2 of the mice developed 2 tumors each,and we kept 1 tumor-bearing mouse for next part experiment).1 of them developed a mammary tumor.Histopathological sections showed 9 cases of salivary gland tumors are SACCs,1 case of mammary tumor was mammary adenocarcinoma.Normal salivary gland,SACC and mammary adenocarcinoma expressed different degrees p63 in the protein level.The c-KIT was not expressed or low expressed in the blank control group,but it was overexpressed in different degrees in SACCs.Mammary adenocarcinoma did not express c-KIT.ConclusionThe TRE-MYB-M.tTA(+/+)mouse model was successfully established,and the salivary gland tumors were SACCs.SACCs formed by TRE-MYB-M.tTA mice all expressed p63 and c-KIT in different degrees.Part IIObjectTo investigate whether MYB overexpression is a cause of the development of SACC in TRE-MYB-M.tTA mice.DesignThe MYB expression of SACCs from Part 1 was detected by Western Blotting and IHC staining.The normal salivary gland tissue and mammary adenocarcinoma were used as controls.The tumor-bearing mouse was fed with R/O water containing2mg/ml doxycycline.We measured the tumor volume every two days.Finally,the tumor tissue was taken for diagnosis and MYB was detected by IHC staining.ResultWestern Blotting showed that 9 cases of SACC and 1 case of mammary adenocarcinoma were overexpressing MYB.IHC results showed that 9 cases of SACC were expressed in different degrees of MYB,no expression in mammary adenocarcinoma.The initial volume of the tumor in the tumor-bearing mouse was4767 mm~3,and its volume significantly reduced over a number of days.The volume was 1368 mm~3 after 83 days.The histopathological section was diagnosed as SACC,and immunohistochemistry showed MYB overexpression but in less cells.ConclusionMYB overexpression caused by MYB gene alteration may be a cause of SACC development.Inhibition the expression of MYB can cause tumor regression.Part IIIObjectTo investigate whether the synergy between MYB and NOTCH-1 promotes the development of SACCs in TRE-MYB-M.tTA(+/+)mice.To preliminarily explore the relationship between SACC and other related tumor pathways and markers.DesignThe expressions of NOTCH-1 of the SACCs from Part I were detected by Western Blotting and IHC staining.The expressions of Hes1,Hes5,Hey1 and Hey2were detected by Real-time PCR at mRNA level.Result9 cases of SACC all overexpressed NOTCH-1 in different degrees in protein level.At the mRNA expression level,the expressions of Hes5,Hey1 and Hey2 in the tumor group were significantly higher than the blank control group(P<0.05).ConclusionThe synergy between MYB and NOTCH-1 may be able to promote the development of SACC in TRE-MYB-M.tTA(+/+)mice.