| 1.Background and Objective Breast cancer is the most commonly malignant tumor in female all over the world.Accumulating researches reveal the mortality of breast cancer is still increasing.The invasiveness and metastasis of breast cancer have been proved to lead the treatment failure.It is significant to discover novel molecular biomarkers as the target for both diagnosis and treatment for breast cancer.The Golgi phosphoprotein 3(GOLPH3)and its interacting proteins are hot spots of tumor researching.GOLPH3 also named GMx33 or GPP34,is a highly conserved 34-k Da trans-Golgi matrix membrane protein,which was known as a key role in receptor recycling,glycosylation and protein trafficking from Golgi to plasma membrane.In recent studies,researchers highlighted the GOLPH3 take the stage not only in the maintenance of trans-Golgi transportation and Golgi morphology,but also as an oncogene in tumorigenesis and migration.There are researches demonstrated oncogene GOLPH3 promotes tumor migration and proliferation by activated m TOR pathway,Wnt pathway,NF-kB pathway,upregulating of MMP-2 and MMP-9 and so on.The results of Qin Zhang and his colleagues’ study revealed the expression of GOLPH3 was associated with tumor migration and proliferation in bladder cancer.The same phenomenon was also found in lung cancer,epithelial ovarian cancer,prostate cancer,hepatocellular carcinoma,rectal cancer and renal cell carcinoma.In recent studies,Zhaolei Zeng and his colleagues also demonstrated GOLPH3 was an oncogene that expressed highly in breast cancer which promotes the migration and proliferation.GOLPH3 is not only an oncogene expressed in tumor,but also the high expression of GOLPH3 indicate a worse prognosis.In the research of Wangfen Tang and his colleagues,they revealed that in the cohort of 100 non-small cell lung cancer patients,52 patients with GOLPH3 high expression illustrated a worse OS than 48 patients with low expression.Ran Wang exhibited the same results by researched the 146 non-small cell lung cancer patients,83 patients with high GOLPH3 expression demonstrated poorer OS than 63 patients with low expression.Xin Hua,Yanling Feng and Qian Li also demonstrated high expression of GOLPH3 correlated with poor OS or DFS in prostate cancer,epithelial ovarian cancer and hepatoma carcinoma cell.Kunli Zhu declared overexression of GOLPH3 correlated a worse DFS and OS for 77 rectal carcinoma patients compared with 70 low expression patients.Furthermore,overexpression of GOLPH3 incurred low sensitivity for neoadjuvant chemotherapy.Their research can be regarded as a unique research of correlation between GOLPH3 and neoadjuvant chemotherapy.Xue Y first noticed 218 renal cell carcinoma patients,high expression of GOLPH3 correlated with poor OS,next they researched the association between GOLPH3 and tumor invasion depth and lymphatic metastasis.The results demonstrated both in the cohort of patients with T1 and T2 or T3 and T4,poor OS was observed in the high expression group.The same phenomenon was showed in both patients with or without lymphatic metastasis.This is the only research noticed the GOLPH3 between tumor invasion depth and lymphatic metastasis.Zhaolei Zeng and his colleagues exhibited overexpression of GOLPH3 incurred the poor OS.There is scarcely ever researches reported how GOLPH3 played its role in breast cancer therapy,thus,increasingly researches are needed to depict the relationship between the expression of GOLPH3 and those clinical features and therapy.In breast cancer therapy,especially,there is no report exhibited the treatment effect of patients with GOLPH3 high expression.In this study,we will investigate the clinical significance of expression of GOLPH3 in breast cancer,detect how potential of migration and proliferation will change after down regulation of GOLPH3 expression in breast cell lines and discover the survival between GOLPH3 expression and different molecular subtype,age,tumor invasion depth,lymphatic metastasis and different surgery type.Vital more,we will discover the response of neoadjuvant chemotherapy and the recurrence and overall survival of patients received adjuvant chemothrerapy with high GOLPH3 expression,aim to find the value of prognosis in breast cancer patients with GOLPH3 high expression,also,find the potential treatment target.2 Methods2.1 Total RNA of brest cancer tissues,ANT,fibroadenoma and cancer cells was extracted by takara reagent.The m RNA expression level of GOLPH3 was assesed by q PCR and protein expression of GOLPH3 was detected by western blotting assays.The correlation of GOLPH3 expression level and patients’ age,tumor size,lymph node metastasis,distant metastasis,molecular sutypies and surgery typies were analyzed.2.2 SiRNA was synthesised to down regulation expression of GOLPH3.QPCR and western blotting assay were used to detected the result of transfection.Migration and invasion abilities of breast cancer cells were detected by transwell migration and invasion assay.The effects of GOLPH3 on breast cancer cells proliferation was detected by CCK-8 assay.The effects of GOLPH3 on breast cancer apoptosis was carried out by flow cytometry.2.3 Overall survival(OS)and disease-free survival(DFS)was assessed by the Kaplan-Meier method and compared by the log-rank test.COX regression models was used for survival data2.4.Chemotherapy treatment54 patients in this cohort of 249 patients received neoadjuvant chemotherapy before surgery.The neoadjuvant chemotherapy strateges were classified into 3 subgroups as follow:22 patients recieved anthracycline+taxane+cyclophosphamide regimen.20 patients received2-5cycles 5-fluorouracil+anthracycline+cyclophosphamide regimen.12 patients received other neoadjuvant chemotherapy regimen.216 patients received adjuvant chemotherapy after surgery.We separated treatments into four ways as follows:74 patients were treated with anthracycline+cyclophosphamide sequential taxane.79 patients recieved5-fluorouracil+anthracycline+cyclophosphamide regimen.The detail was demonstrated above.30 patients underwent 4-6 cycles taxane+cyclophosphamide regimen.33 patients received other adjuvant chemotherapy regimen.2.5 Statistical analysis All the statistical analyses were performed with SPSS Statistics 21.0(IBM,Chicago,USA)and Graphpad Prism6.0.Comparisons between groups for statistical significance were carried out with a 2-tailed paired Student t test.The relationship between the expression of GOLPH3 and clinicopathological characteristics was evaluated by Chi-square test,Fisher’s exact test was used when the unit was less than six.Overall survival(OS)and disease-free survival(DFS)was assessed by the Kaplan-Meier method and compared by the log-rank test.COX regression models was used for survival data.P value less than 0.05 was considered statistically significant.3 Results3.1 Expression of GOLPH3 in tissue samples and cell linesThe results of immunohistochemistry and western blotting illustrated that compared with ANT and fibroadenomas,breast cancer tissues expressed higher level of GOLPH3(p < 0.05).The m RNA expression in tissues analysised by q PCR exhibited the same consequence as the results demonstrated by immunohistochemistry and western blotting.According to the results of immunohistochemistry,the expression of GOLPH3 can be classified into +,++and +++.Moreover,according to the TNM classification,the expression of GOLPH3 exhibited a trend that the quantity of GOLPH3 expressing increasing as the TNM classification increasing.The m RNA and protein expression of GOLPH3 in cell lines were assessed by q PCR and western blotting assays,respectively.The results demonstrated that GOLPH3 expressed in both breast cancer cell lines,moreover,the cell line MDA-MB-231,with high potential of invasiveness expressed higher GOLPH3 than the MCF-7,which with lower potential of invasiveness.3.2 Silencing of GOLPH3 expression reduces the potential of migration,invasion,proliferation,and promotes the apoptosis in breast cancer cellsAfter antibiotic selection,the downregulation of GOLPH3 by siRNA was confirmed by q PCR and western blot.The ability of migration and invasion was reduced in GOLPH3-si RNA compared with parent cell lines and scram-si RNA(p < 0.05).Compared with parent cell lines and scram-si RNA groups,the GOLPH3-si RNA group demonstrated that after the expression of GOLPH3 was down regulated,the proliferation ability was reduced as well.It is indicated that knocked down of GOLPH3 induced the apoptosis of breast cancer cells.3.3 Overexpression of GOLPH3 is associated with worse survival in breast cancer patientsThe results highlight that the expression of GOLPH3 has tight correlation with T classification(p=0.033),N classification(p=0.000),M classification(p=0.000),molecular subtype(p=0.012)and surgery type(p=0.000).It is obviously indicated that compared with low expression,high expression of GOLPH3 exhibited worse DFS and OS in breast cancer patients.The results of single factor survival analysis and multiple factor analysis revealed that GOLPH3 was important prognosis factors for DFS and OS of breast cancer patients.Molecular subtype of breast cancer was regarded has correlation with GOLPH3(p=0.012).Our study also illustrated overexpression of GOLPH3 incurs poor DFS and OS in every single type of all molecular subtype.However,when focused the cohort of patients with GOLPH3 low expression,the comparation between five molecular subtyps exhibited no statistical significant both for DFS and OS(DFS:p=0.7547,OS:p=0.4734)it is the same phenomenon showed in the cohort of patients with high expression of GOLPH3(DFS:p=0.6979,OS:p=0.6527).3.4 The association between age,tumor invasion depth,lymphatic metastasis and survival of breast cancer patients with GOLPH3 expressionThe results highlight in the cohort of patients age<40,compares with low expression,the overexpression of GOLPH3 won’t increase the risk of recurrence in breast cancer(p=0.5659),but the OS seems significantly poorer for GOLPH3 high expression rather than low expression(p=0.0187).For other all age bracket patients,worse DFS and OS was obvious visible in GOLPH3 high expression compared with low expression.In the comparation of four groups,there is no statistical significant in DFS and OS of patients with GOLPH3 low expression.The risk of recurrence is increasing as the age increasing in the group of overexpression GOLPH3.In the comparation of four groups,there is no statistical significant in OS of patients with GOLPH3 high expression.Tumor invasion depth has tight relationship with GOLPH3(p=0.033).To affirm the survival of GOLPH3 expression and tumor invasion depth,we separated the patients from T1 to T4 according to T classification.For those patients of T1 and T2,high GOLPH3 expression lead to poorer both DFS and OS rather than low GOLPH3 expression(T1:DFS:p=0.0103,OS:p=0.0101,T2:DFS:p<0.0001,OS:p<0.0001).However,there is no statistical significance of DFS and OS between GOLPH3 high and low expression in patients of T3 and T4classification(T3:DFS:p=0.1389,OS:p=0.1270,T4:DFS:p=0.1203,OS:p=0.4716).For patients with high GOLPH3 expression,T3 and T4 classification indicate poor DFS(p=0.0360)but not OS(p=0.7521).For patients with low GOLPH3 expression,the increasing of tumor invasion depth means worse DFS(p=0.0186)and OS(p=0.0222).The relationship of GOLPH3 and lymphatic metastasis was observed in our study(p<0.001).For patients without lymphatic metastasis(N0),high GOLPH3 indicate poor DFS(p=0.0004)and OS(p=0.0166).However,for patients with lymphatic metastasis(N1 and N2),the expression of GOLPH3 seems has no influence with DFS and OS(N1:DFS:p=0.3957,OS:p=0.6825,N2:DFS:p=0.4373,OS:p=0.6775).In the cohort of people with GOLPH3 low expression,the increasing of lymphatic metastasis correlates with poorer DFS(p< 0.0001)rather than OS(p=0.3053).While,in the cohort of people with high GOLPH3 expression,the increasing of lymphatic metastasis illustrated worse DFS(p=0.0012)and OS(p<0.0001).3.5 Surgery type will no influence the survival of patients with GOLPH3 high expressionWe noticed those patients received breast conserving surgery,simple mastectomy,modified radical mastectomy and section resection,respectively.In the cohort of patients received breast conserving surgery,there were 17 patients exhibited GOLPH3 high expression and 1 low expression.Because of the small sample,the analysis of DFS and OS couldn’t reveal the real influence imposed by GOLPH3 high expression for the patients received breast conserving surgery.The same phenomenon can be observed in the cohort of patients received simple mastectomy and section resection.In the cohort of patients received modified radical mastectomy,high expression of GOLPH3 induced poorer DFS and OS than low expression(p < 0.0001).While,for those patients with GOLPH3 low expression,the results of DFS and OS showed no statistical significance by compared of four surgery types.In the cohort of patients with GOPH3 high expression,although,the results of DFS and OS showed there was a statistical significance by compared of four surgery types(DFS:p=0.0240,OS :p=0.0182),but we considered as the results was analyzed by a small sample research,so the occasionality couldn’t be avoided.3.6 High expression of GOLPH3 correlated with neoadjuvent chemotherapy resistance in breast cancer patientsIn our research,54 patients received neoadjuvant chemotherapy.36 patients with high expression of GOLPH3 exhibited lower PR rate than 18 patients with low GOLPH3 expression(p=0.021).In the cohort of 54 patients,22 of them received anthracycline+taxane+cyclophosphamide regimen,20 patients received5-fluorouracil+anthracycline+cyclophosphamide regimen and 12 received other regimen.In the group underwent anthracycline+taxane+cyclophosphamide regimen,14 patients with high GOLPH3 expression revealed lower PR rates(6/14)than 8 patients with low GOLPH3 expression(7/8)(p=0.05).In the group of patients underwent 5-fluorouracil+anthracycline+cyclophosphamide and other regimens,the PR rates demonstrated no statistical significance between GOLPH3 high expression and low expression.(p=0.492,p=0.296).3.7 High expression of GOLPH3 correlated with high recurrence rate in breast cancer patients received adjuvant chemotherapy216 patients received adjuvant chemotherapy after surgery.For these 216 patients,103 with GOLPH3 high expression correlated with poorer DFS and OS than 113 with low expression(DFS:p<0.0001,OS:p<0.0001).In this cohort of216 patients,74 of them received anthracycline+cyclophosphamide sequential taxane regimen,patients with high GOLPH3 reveals easier recurrence(p=0.035)and poor OS(p=0.0015).79 patients received 5-fluorouracil+anthracycline+cyclophosphamide,the same recurrence(p=0.010)and OS(p=0.0017)was observed as patients received anthracycline+cyclophosphamide sequential taxane regimen(Table4 and Figure2 I).While,in the group of 30 people received taxane+cyclophosphamide regimen,the results of recurrence exhibited no statistical significance between high GOLPH3 and low(p=0.190),also we obtained the same conclusion in overall survival(p=0.2129).For those patients received other chemotherapy regimens,overexpression of GOLPH3 indicated high risk of recurrence(p=0.027)but not poorer overall survival(p=0.0845).4 Conclusions4.1 GOLPH3 expressed higher in breast cancer tissues rather than ANT and fibroadenoma.The expression of GOLPH3 has tight correlation with T classification,N classification,M classification,molecular subtipes and surgery types.4.2 Down regulation of GOLPH3 expression inhibited the ability of migration,invasion and proliferation of breast cancer cells.Down regulation of GOLPH3 expression enhanced the apoptosis of breast cancer cells.4.3 Overexpression of GOLPH3 correlated with poor survival of breast cancer patients.GOLPH3 is the oncogene with potential of prognosis.The results highlight in the cohort of patients age<40,compares with low expression,the overexpression of GOLPH3 won’t increase the risk of recurrence in breast cancer,but the OS seems significantly poorer for GOLPH3 high expression rather than low expression.For other all age bracket patients,worse DFS and OS was obvious visible in GOLPH3 high expression compared with low expression.For those patients of T1 and T2,high GOLPH3 expression lead to poorer both DFS and OS rather than low GOLPH3 expression..For patients with high GOLPH3 expression,T3 and T4 classification indicate poor DFS but not OS.For patients with low GOLPH3 expression,the increasing of tumor invasion depth means worse DFS.For patients without lymphatic metastasis(N0),high GOLPH3 indicate poor DFS and OS.However,for patients with lymphatic metastasis(N1 and N2),the expression of GOLPH3 seems has no influence with DFS and OS.In the cohort of people with GOLPH3 low expression,the increasing of lymphatic metastasis correlates with poorer DFS rather than OS.While,in the cohort of people with high GOLPH3 expression,the increasing of lymphatic metastasis illustrated worse DFS and OS.4.4 patients with high expression of GOLPH3 exhibited lower PR rate than patients with low GOLPH3 expression.In the group underwent anthracycline+taxane+cyclophosphamide regimen,patients with high GOLPH3 expression revealed lower PR rates than patients with low GOLPH3 expression.For patients received adjuvant chemotherapy after surgery,patients with GOLPH3 high expression correlated with poorer DFS and OS than low expression.Innovation1.We discover the survival between GOLPH3 expression and different molecular subtype,age,tumor invasion depth,lymphatic metastasis and different surgery type.There is no research reported the results.2.We discovered the response of neoadjuvant chemotherapy and the recurrence and overall survival of patients received adjuvant chemothrerapy with different GOLPH3 expression.There is no research reported the results. |
PDF Full Text Request